Topical application of CRISPR-modified bacteria to treat acne vulgaris

ABSTRACT

Compositions, systems and methods of improving the health of the microbiome of an individual&#39;s skin relate to the provision of skin contacting formulations containing beneficial bacteria and other microbe components to foster the growth and maintenance of a healthy skin microbiome. Embodiments include methods for treating an individual suffering from acne vulgaris by topically administering a composition that includes live bacteria selected from the group consisting of  L. reuteri, L. johnsonii, L. crispatus, C. acnes , and  Nitrosomonas eutropha , that have been modified by using a using a clustered regularly interspaced short palindromic repeats (CRISPR) CRISPR associated protein (Cas) system or a CRISPR from  Prevotella  and  Francisella  1(Cpf1) system to reduce the production of a virulence factor of the bacteria.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 18/087,545, filed Dec. 22, 2022), which is acontinuation-in-part of U.S. patent application Ser. No. 17/854,422,filed Jun. 30, 2022, which is a continuation-in-part of U.S. patentapplication Ser. No. 17/848,759, filed Jun. 24, 2022, which is acontinuation-in-part of U.S. patent application Ser. No. 17/835,204filed Jun. 8, 2022 (now U.S. Pat. No. 11,529,379, issued Dec. 20, 2022),which is a continuation-in-part of U.S. patent application Ser. No.17/567,295 filed Jan. 3, 2022, which is a continuation-in-part of U.S.patent application Ser. No. 17/337,600, filed Jun. 3, 2021 (now U.S.Pat. No. 11,213,552, issued Jan. 4, 2022), which is acontinuation-in-part of Ser. No. 17/027,953, filed on Sep. 22, 2020 (nowU.S. Pat. No. 11,026,982, issued Jun. 8, 2021), which is acontinuation-in-part of U.S. patent application Ser. No. 16/917,096,filed Jun. 30, 2020 (now U.S. Pat. No. 10,940,169, issued Mar. 9, 2021),which is a continuation-in-part of U.S. patent application Ser. No.16/782,364, filed Feb. 5, 2020 (now U.S. Pat. No. 10,835,560, issuedNov. 17, 2020), which is a continuation-in-part of U.S. patentapplication Ser. No. 16/423,375, filed May 28, 2019 (now U.S. Pat. No.10,555,976, issued Feb. 11, 2020), which is a continuation of U.S.patent application Ser. No. 16/160,336, filed Oct. 15, 2018 (now U.S.Pat. No. 10,314,866, issued Jun. 11, 2019), which is a continuation ofU.S. patent application Ser. No. 15/403,823, filed Jan. 11, 2017 (nowU.S. Pat. No. 10,111,913, issued Oct. 30, 2018), which is anon-provisional of U.S. Provisional Patent Application Ser. No.62/296,186, filed on Feb. 17, 2016.

This application is a continuation-in-part of U.S. patent applicationSer. No. 16/426,346, filed May 30, 2019 (now U.S. Pat. No. 10,716,815,issued Jul. 21, 2020), which is a continuation of U.S. patentapplication Ser. No. 15/639,767, filed Jun. 30, 2017 (now issued U.S.Pat. No. 10,314,865, issuing Jun. 11, 2019), which is acontinuation-in-part of U.S. patent application Ser. No. 15/437,976,filed Feb. 21, 2017 (now U.S. Pat. No. 9,730,967, issued Aug. 15, 2017),which is a continuation-in-part application of U.S. patent applicationSer. No. 15/228,454, filed Aug. 4, 2016 (now U.S. Pat. No. 9,585,920,issued Mar. 7, 2017), which is a continuation-in-part application ofU.S. patent application Ser. No. 14/954,074, filed on Nov. 30, 2015 (nowissued U.S. Pat. No. 9,457,077, issued Oct. 4, 2016).

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This application is a continuation-in-part application of U.S. patentapplication Ser. No. 16/037,053, filed Jul. 17, 2018 (abandoned).

The present application is a continuation-in-part of U.S. patentapplication Ser. No. 17/543,992, filed Dec. 7, 2021, which is acontinuation-in-part of U.S. patent application Ser. No. 16/804,361,filed Feb. 28, 2020 (now U.S. Pat. No. 11,191,665, issued Dec. 7, 2021),which is a continuation-in-part of U.S. patent application Ser. No.16/020,433, filed Jun. 27, 2018 (now U.S. Pat. No. 10,583,033, issuedMar. 10, 2020), which is a continuation-in-part application of U.S. Ser.No. 15/342,642, filed Nov. 3, 2016 (now U.S. Pat. No. 10,010,568, issuedJul. 3, 2018), which seeks priority from U.S. Provisional PatentApplication Ser. No. 62/260,906, filed Nov. 30, 2015.

The present application is a continuation-in-part of U.S. Ser. No.15/270,034, filed Sep. 20, 2016 (now U.S. Pat. No. 9,750,802, issuedSep. 5, 2017), which is a continuation-in-part of U.S. patentapplication Ser. No. 14/954,074, filed Nov. 30, 2015 (now U.S. Pat. No.9,457,077, issued Oct. 4, 2016), which is a continuation-in-part of U.S.patent application Ser. No. 14/574,517, filed Dec. 18, 2014, (now U.S.Pat. No. 9,408,880, issued Aug. 9, 2016), which claims priority fromU.S. Provisional Patent Application Ser. No. 62/072,476, filed on Oct.30, 2014, U.S. Provisional Patent Application Ser. No. 62/053,926, filedSep. 23, 2014, U.S. Provisional Patent Application Ser. No. 62/014,855,filed Jun. 20, 2014 and U.S. Provisional Patent Application Ser. No.61/919,297, filed on Dec. 20, 2013.

This application is also a continuation-in-part of U.S. patentapplication Ser. No. 17/836,079, filed Jun. 9, 2022, which is acontinuation-in-part of U.S. patent application Ser. No. 16/884,772filed on May 27, 2020 (now U.S. Pat. No. 11,357,722, issued Jun. 14,2022, which is a continuation-in-part of U.S. patent application Ser.No. 16/136,950, filed Sep. 20, 2018 (now U.S. Pat. No. 10,668,014,issued Jun. 2, 2020), which is a continuation of U.S. patent applicationSer. No. 15/385,278, filed Dec. 20, 2016 (now U.S. Pat. No. 10,085,938,issued Dec. 2, 2018).

This application is also a continuation-in-part of U.S. patentapplication Ser. No. 14/752,192 filed on Jun. 26, 2015 (now U.S. Pat.No. 9,549,842, issued Jan. 24, 2017).

The entire disclosure of the prior applications are considered to bepart of the disclosure of the accompanying application and are herebyincorporated by reference.

FIELD OF THE INVENTION

Compositions, systems and methods of improving the health of themicrobiome of an individual's skin relate to the provision of skincontacting formulations containing beneficial bacteria and other microbecomponents to foster the growth and maintenance of a healthy skinmicrobiome.

BACKGROUND OF THE INVENTION

The skin is the human body's largest organ, colonized by a diversemilieu of microorganisms. Colonization is driven by the ecology of theskin surface, which is highly variable depending on topographicallocation, endogenous host factors and exogenous environmental factors.Microorganisms including bacteria, fungi, and viruses are known tocolonize the skin. Human skin continuously undergoes self-renewal, soresident microbial cells are shed in the process. Most of the microbesfound on the skin are harmless to healthy individuals. Some areconsidered to be mutualistic organisms and confer health benefits to theskin by secreting, for example, antibacterial substances, preventingpathogen colonization, and influencing host immune responses. Changes inskin ecology or microecology with an excessive proliferation ofpathobionts results in a spectrum of skin diseases from mild acne, todermatitis and psoriasis. Further, skin pathobionts may accelerate theprogression of skin diseases when the skin barrier is breached. On theother hand, commensal microorganisms can cause diseases and infectionsif the physical barrier of the skin has been compromised due to traumaor injuries. Changes in skin ecology or microecology with an excessiveproliferation of pathobionts results in a spectrum of skin diseases frommild acne, to dermatitis and psoriasis. Skin pathobionts may acceleratethe progression of skin diseases when the skin barrier is breached.

The skin and gastrointestinal (“GI”) tracts of humans are colonized by adiverse array of microorganisms beginning at the time of birth when aninfant is exposed to the maternal microflora and other environmentalmicrobes. From the time of initial colonization, the human microbiomeremains in a state of flux where the composition of the residentmicroflora changes over time in response to factors intrinsic andextrinsic to the host.

Probiotics are so-called “good” microorganisms (typically bacteria) thatare ingested (or contacted with a person) alive by an individual so thatthe introduced microorganisms can colonize the GI tract of the person.Conventional prebiotics are ingestible ingredients that selectivelysupport the growth or survival of the “good” microorganisms which aredesirably present in the GI tract. Conventional prebiotics are typicallya nutrient source (e.g., fructooligosaccharide orgalactooligosaccharide) that can be assimilated by one or more membersof the GI microbiome, but which are not digestible by the human host.

Human skin is colonized by a diverse array of microorganisms, with suchcolonization beginning shortly after birth when an infant is exposed tothe maternal microflora. From the time of initial colonization, thehuman microbiome changes over time in response to factors intrinsic andextrinsic to the host. The makeup of the human skin microbiome differssignificantly from the makeup of the GI microbiome in terms of both thetype and variety of microorganisms present.

Members of the GI and skin microbiomes utilize different nutrientsources due to, at least in part, the starkly contrasting environmentsin which the two microbiomes are found and the substrates available foruse as food. Dietary requirements of microorganisms can varysignificantly from one species to the next, and it is not uncommon foran agent that exhibits prebiotic activity on a particular microorganismto exhibit no prebiotic activity on a different microorganism. Forexample, prebiotics designed for the GI microbiota have historicallybeen carbohydrate-based materials that serve as food for residentglycolytic driven microorganisms. The microflora present on the skin ofa person, however, can include lipophilic organisms, which would notnecessarily be expected to assimilate carbohydrates. Even the glycolyticmicroorganisms present on the skin may not utilize the same kinds ofcarbohydrates as GI microbes. The make-up of the GI and skin microbiomesof a human may vary significantly and there can also be significantvariability in the make-up of the same microbiome between individuals.The surface of mammalian skin typically includes a wide variety ofmicroorganisms, which may vary from species to species, individual toindividual, and from location to location on an individual. Certainundesirable microorganisms, such as pathogenic bacteria, yeasts andmolds, may attempt to colonize the skin and upset the balance of ahealthy microbiome.

The development of molecular techniques to identify and quantifymicrobial organisms has revolutionized the microbial world. Genomiccharacterization of bacterial diversity relies on sequence analysis ofthe 16S ribosomal RNA gene, which is present in all bacteria andarchaea. The 16S rRNA gene contains species-specific hypervariableregions, which allow taxonomic classification, and highly conservedregions, which act as a molecular clock and a binding site for PCRprimers. Using current technologies, an organism does not need to becultured to determine its type by 16S rRNA sequencing.

The global population is rapidly aging. Currently, 566 million peopleare at least 65 years old worldwide, with estimates of nearly 1.5billion by 2050, particularly in developing countries. Infectionsconstitute a third of mortality in people at least 65 years old.Moreover, lengthening life spans correlate with increased time inhospitals or long-term care facilities and exposure to drug-resistantpathogens. The risk of nosocomial infections increases with age,independent of duration spent in healthcare facilities. One theory isthat as a person ages, their immune system changes and is less robust inaddressing bacterial infections. By enhancing the microbiome of aperson's skin as they age, it is believed that infections that wouldotherwise be encountered will be avoided, or at least the frequency andseverity of the same will be decreased.

Acne affects hundreds of millions of people worldwide, believed toinvolve the bacterial colonization of affected skin and consequentinflammation. Acne leads to emotional distress and even depression.Sebum is produced by the skin's sebaceous glands, providing nutrientsfor a number of bacteria, including the anaerobic gram-positivebacterium Cutibacterium acnes, which decomposes the sebum fats toglycerine and fatty acids. This often results in an inflammation of theskin and the formation of pimples and pustules. As used herein, theterms Propionibacterium acnes and Cutibacterium acnes should beunderstood to refer to the same bacteria and can be usedinterchangeably. Topical applications of antibiotics, includingerythromycin, clindamycin, metronidazole, sulfacetamide, doxycycline orminocycline, or systemic applications of antibiotics, can be used toreduce the number of C. acnes, but there are high relapse rates andissues due to development of antibiotic-resistant bacteria.

C. acnes is considered an aerotolerant anaerobe because it possessesenzymatic systems able to detoxify oxygen, allowing it to be sustainedon the surface of the skin.

The genus C. acnes has been further subdivided into subspecies, such asC. acnes subsp. defendens and C. acnes subsp. elongatum. Unlike otherGram-positive bacteria, C. acnes has a unique cell wall and envelope,containing phosphatidylinositol, triacylglycerol, and many other commonlipids. The cell wall of C. acnes consists of peptidoglycan (PNG), butof a type different from that of other Gram-positive bacteria, in thatthe peptide chain contains the L-acid L-diaminopelic acid and D-alanine.C. acnes has a strong impact of the skin's immune system.

There is a long-felt need for effective treatments to enhance the healthof an individual's skin. The present invention provides a method andsystem for satisfying such a need.

SUMMARY OF THE INVENTION

One aspect of the present invention is directed to the use of humanspecific species of bacteria that are then modified to enhance one ormore characteristics deemed beneficial to the skin microbiome and healthof the individual, including bacteria that have been modified via aCRISPR-Cas9 and/or Cpf1 system to either repress the expression of aparticular protein or lipid, or to increase the production of beneficialmicrobial secretions. Clustered Regularly Interspaced Short PalindromicRepeats from Prevotella and Francisella 1 or CRISPR/Cpf1 is aDNA-editing technology analogous to the CRISPR/Cas9 system. Oneobjective is to avoid modifying an individual's human genome, butinstead, to significantly affect the health of humans by employingmodifications to the skin microbiome. Use of human specific strains ofbacteria, whether they are commensal or pathogenic, including bacteriathat are modified to alter their native pathogenicity, is one preferredaspect of many embodiments of the present invention.

Certain aspects of the present invention are directed to a method foraltering the microbiome of an individual's skin by administering to aregion of the skin of an individual an effective amount of a bacterialformulation. In one preferred embodiment, the individual is a newbornand the step of administering is performed within the first 6 hours ofthe newborn's birth. Such a bacterial formulation may be a lotion,ointment or gel adapted to be rubbed onto the newborn's skin. Thebacteria included in the bacterial formulation may vary to addressparticular concerns or diseases. For example, the bacterial formulationmay include bacteria selected from the group consisting of Nitrosomonaseutropha and Propionibacterium species. More particularly, theequilibrium of a bacterial population of the region of the skin of theindividual is modified to increase the number of Propionibacteriumbacteria and to decrease the number of Staphylococcus bacteria on theindividual's skin in such region. In other embodiments, the bacterialformulation includes the bacteria Staphylococcus aureus that has beenmodified by employing a CRISPR-Cas or Cpf1 system to interfere with S.aureus virulence regulation involving the Agr quorum-sensing signalingmolecule. In several embodiments, the bacterial formulation comprises abacteria that has a tropism specific for the human species. In others,the bacterial formulation comprises at least two of the bacteriaselected from the group consisting of: Prevotella, Lactobacillusjohnsonii, Bacteroides fragilis, Lactobacillus ruminis and L. infantis.In certain embodiments the bacteria is an ammonia oxidizing bacteria. Inother embodiments, the region of the skin to which the bacterialformulation is applied is the scalp. In various embodiments, rather thanusing a wild-type bacteria, the bacteria employed is one that has beenmodified by CRISPR-Cas or CRISPR-Cpf1 to delete a functional virulencefactor from the bacteria. In particular embodiments, the method includesadministering to the skin a bacteria that produces tomatidine. Inothers, the bacteria produces p53. Thus, in some embodiments, the methodinvolves use of bacteria wherein a CRISPR-Cas or CRISPR-Cpf1 system isemployed to insert a gene for the production of tomatidine and/or p53into at least one of the bacteria in the bacterial formulation. Inothers, a CRISPR-Cas or CRISPR-Cpf1 system is employed to insert one ormore genes into the bacteria comprising the bacterial formulation tofacilitate the oxidizing of ammonia by the bacteria. To further enhancethe ability of desired bacteria to be maintained on the skin of anindividual, certain methods further comprise administering to theindividual's skin a prebiotic that comprises a nutrient source for thebacteria that is assimilated by the bacteria, and preferably one that isnot digestible by the individual. In particular embodiments, the methodfurther includes administering to the skin an extract derived from ahelminth selected from the group consisting of Capillaria hepatica,Dicrocoelium dendriticum, Ascaris lumbricoides, Enterobius vermicularis,Trichuris trichiura, Ancylostoma duodenale, Necator americanus,Strongyloides stercoralis, Haemonchus contortus, and Trichinellaspiralis. In still others, the bacterial formulation includes at leastone arabinogalactan. Yet others include at least one of the following:L. infantis, and L. johnsonii. In a particular embodiment, the bacterialformulation includes at least one bacteria modified via a CRISPR-Cassystem to express a gene encoding interferon regulatory factor 4.

In particular embodiments, in view of the tropism demonstrated by S.pyogenes for humans, and the recognition that such bacterial species isfound in both the oral and skin microbiome of humans, S. pyogenes is apreferred bacterial species to employ in various embodiments of thepresent invention.

In still other embodiments, the focus is on interspecies interactionswithin mixed microbial communities, with the objective being to modifycompetitive relationships involving nonbiocidal biosurfactants, enzymes,and metabolites produced by bacteria and other microorganisms in amanner such that selection of particular bacterial species can beemployed to inhibit initial adhesion, trigger matrix degradation,encourage jamming of cell-cell communications, and induce biofilmdispersion. Nonbiocidal molecules are thus employed to modifycompetitive interactions within biofilms in a manner that promotes theoverall health of an individual's microbiome, especially on the skin.

In certain embodiments, a bacterial formulation is applied to newbornswithin a critical window of time after birth, preferably within thefirst 24 hours of the newborn's birth, more preferably within 6 hours oftheir birth, even more preferably within 3 hours of birth, and mostpreferably within an hour after their birth. The administration can beby several methods, but preferably is a lotion, ointment or gel that isrubbed onto the newborn's skin, preferably all over his/her entire body.A spray or mist can also be applied that contains the bacterial andmicrobe formulations as set forth herein. While not bound by theory, thecritical window to apply to the newborn's skin the referencedformulations, e.g., microbial mixtures of bacteria beneficial intriggering immune system development, is within a relatively short timeperiod and is necessary to establish immune tolerance to a variety ofcommensal microbes. The way and content of microbes presented at a timein which a newborn has his/her skin colonized establishes immunetolerance to particular commensal microbes. The influx of highlyactivated T cells into neonatal skin is believed to occur in suchcritical window. So a mother of a newborn has a choice: to simply relyupon chance as to what particular microbes might be present during thiscritical window of the newborn's establishing immune tolerance toparticular bacteria and other microbes; or to provide the newborn with aselected formulation containing predetermined microbes such that thenewborn's developing immune system can properly react to the microbes inthe predetermined formulation, and thus provide the newborn with theopportunity to develop a more expansive immune tolerance profile. It isbelieved that the mechanism that promotes tolerance is tissue specific,and thus, the skin and the gut may have different ways by which theymediate tolerance to commensal microbes. To establish a healthy statusof a newborn's skin as it relates to commensal microbes on its skin, theparticular type of microbes, including bacteria, brought into contactwith his/her skin is achieved in a certain time period after birth (e.g.within 1 to 24 hours after birth) so that the developing immune systemof the infant establishes tolerance to such microbes, thus avoidingallergies, autoimmune diseases and other related diseases, as well aschronic inflammation of the skin.

In certain embodiments of the present invention, the skin microbiome isenhanced via providing microbes able to metabolize lipids, proteins andcarbohydrates, and thus, produce acid that aids in maintaining theso-called “acid mantel” of the skin. In preferred embodiments thebacteria that is modified has a very narrow host tropism, such that thebacteria are specific for the human species and thus, their modificationposes little if any risk to other animals or organisms.

Other embodiments are directed to combating infections of a person'sskin by the bacteria Staphylococcus aureus. Staphylococcus aureus is acommensal and pathogen of both humans and cattle. In certain embodimentsthe accessory gene regulator (Agr) system and the virulence regulationof S. aureus pathogenesis is modified to delete or to at least reducethe virulence of the bacteria. In such a way, the present inventionprovides a way to effectively combat S. aureus infections. In variousembodiments of the present invention, CRISPR-Cas9 and/or Cpf1 systemsare employed to render ineffective virulence factors of such bacteriainvolved with the establishment and propagation of infection. Severalmolecules have been found to interfere with S. aureus virulenceregulation, especially those targeting the Agr quorum-sensing signalingmolecule. By modification of this bacterial species using CRISPR-Casand/or Cpf1 it is possible to achieve broad-spectrum inhibitory effectson most S. aureus strains and Agr subtypes.

The tropism of individual bacteria for particular host tissues (e.g.,skin vs. respiratory tract vs. gastrointestinal tract) is determined bythe array of available adhesion-receptor pairs. In preferredembodiments, bacteria having substantial, if not entire, human hostspecificity are employed. For example, Salmonella enterica serovartyphi, known to be the bacteria responsible for typhoid fever, alife-threatening human disease, demonstrates strict human hostspecificity. In certain embodiments, the virulence factors of suchbacteria are compromised by being modified via the CRISPR-Cas or Cpf1system to render the modified bacteria as non-pathogenic. Similarly, thebacteria Neisseria, the causative agent of gonorrhea, is a diseaserestricted to humans, and thus similar CRISPR-Cas and/or Cpf1 systemsmay be employed to reduce if not eliminate the virulence factors of suchbacteria. Likewise, Helicobacter pylori is known to be an etiologicagent of gastritis and peptic ulcer disease in humans. The ironacquisition system of H. pylori by the human lactoferrin receptor systemis believed to play a major role in the virulence of H. pyloriinfection. The CRISPR-Cas and/or Cpf1 systems may be employed to reduceif not eliminate the virulence factors of this bacteria. Yet anotherbacteria demonstrating human tropism is Haemophilus influenzae, a Gramnegative species that requires heme and has exclusive human hostspecificity. In certain embodiments, the CRISPR-Cas and/or Cpf1 systemsmay be employed to reduce if not eliminate the virulence factors of suchbacteria. The distinction between throat and skin group A Streptococcushas become blurred and to date there have been few advances in treatmentof group A Streptococcus skin infections. Certain aspects of the presentinvention include the modification of skin group A Streptococcus toreduce the likelihood, if not prevent, related skin diseases, includingeczema, atopic dermatitis, acne, allergic inflammation, skinhypersensitivity, UV-induced skin damage, and skin cancer.

One particular aspect of certain embodiments of the present inventionrelates to the treatment of acne. Acne is the most common skin diseaseaccounting for a quarter of dermatologists' patient volume. Acne is achronic disease that can significantly impact an individual's quality oflife with social, psychological and emotional impairments. Thus, invarious embodiments, bacteria are selected that, once applied to anindividual's skin, is able to ameliorate acne. Such bacteria includeammonia oxidizing bacteria, preferably provided to a person's skin incombination with a pharmaceutically acceptable excipient. In certainembodiments, bacteria are employed to achieve topical nitric oxiderelease at or near the surface of the skin and addition of urea orammonium salts to the skin provides additional substrates that thesebacteria utilize to form nitrite. While not intending to be limitedthereby, such ammonia oxidizing bacteria may be selected from the groupconsisting of Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosocystis,Nitrosolobus, Nitrosovibrio, and combinations thereof. In someinstances, the ammonia oxidizing bacteria is Nitrosomonas eutropha (N.eutropha). Such ammonia-oxidizing bacteria are employed to improve skinhealth and are able to convert ammonia to nitrite, an antimicrobialcompound, and nitric oxide. Various aspects of the present invention aredirected at restoring and maintaining the delicate balance of the skinmicrobiome.

The present invention in various embodiments is directed to a variety ofconsumer products including cosmetic products such as skin care products(bath preparations, skin washing and cleaning products, skin careproducts, eye cosmetics, lip care products, nail care products, intimatehygiene preparations, foot care), those with special effects(sunscreens, tanning agents, deodorants, anticholinergics, depilatories,shaving, fragrance), those for oral or dental hygiene and those for haircare (shampoos, conditioners, etc.)

One objective of the present invention is to achieve various health andcosmetic benefits by providing a healthy, balanced skin microbiome.Modified bacteria that are beneficial to the skin, especially thosemodified using CRISPR-Cas systems, are used to enhance the beneficialcharacteristics of skin microbiomes in a manner that purposefullyexposes skin to microbes, rather than the conventional use ofanti-bacterial agents to kill bacteria—including beneficial bacteria—ona person's skin. The adherence to the skin of problem flora, such aspathogenic bacteria and yeast, has been associated with numerousailments, including skin infections, diaper rash, urinary or vaginalinfections, and malodors. Use of the present invention addresses suchissues in a novel and non-obvious manner.

Other embodiments are directed to prebiotic agents for use on skin. Inpreferred embodiments, CRISPR-Cas and/or Cpf1 modified bacteria,especially those demonstrating total or substantial tropism for humans,are employed in one or more of the above referenced products, withcertain features, namely, virulence factors reduced if not eliminated.In such a manner, there is a competitive inhibition of undesiredbacteria with the modified bacteria as set forth herein. In certainembodiments, the cleansing of one's skin to effectively reduce by atleast about 50%, more preferably about 30%, and most preferably toreduce by at least about 25%, of native bacteria on an individual's skinportion to be addressed, is performed prior to purposefully contactingthe individual's skin with one or more bacteria, and in particular,bacterial species that have been modified via employment of a CRISPR-Casand/or Cpf1 system to reduce if not effectively compromise the virulencefactors of such bacteria, and more preferably a bacteria that has a hostspecificity exclusive to humans.

In one particular embodiment, bacteria are modified via a CRISPR-Cassystem to express a gene identified for grey hair—interferon regulatoryfactor 4 (IRF4). This gene is involved in regulating production andstorage of melanin, the pigment that determines hair, skin and eyecolor. Hair greying is caused by an absence of melanin in hair. Thus, onvarious embodiments, bacteria are modified to express IRF4 and topicalapplication of such bacteria to an individual's scalp provides for theprevention of hair turning grey as it otherwise would without suchapplication of such bacteria. In still other embodiments, bacteria aremodified to express levels of melanin to maintain hair color when suchmodified bacteria are contacted with the scalp of an individual.

Certain aspects of the present invention are directed to topicallyapplying live probiotic lactobacilli to beneficially modulate cutaneousmicrobial interactions and host inflammatory responses in individualswith skin diseases and conditions, including acne symptoms. In otherembodiments, postbiotic formulations derived from Lactobacilli—which mayinclude bacterial lysates, supernatants, and/or purified supernatants ormetabolites, are applied to an individual's skin.

The skin microbiome also includes various Lactobacillus taxa—includingthose known to be dominant in the human vagina. One aspect of certainembodiments of the present invention is directed to the use of bacteriaon an individual's skin that many associate solely with the vaginalmicrobiome. For example, preferably one of Lactobacillus crispatus, L.iners, L. gasseri and L. jensenii are employed, most preferably withsuch bacteria being modified via use of a CRISPR system to reduce oreliminate one or more virulence factors. Other embodiments also includeone or more of the following: L. plantarum/L. pentosus group and L.casei/paracasei/rhamnosus group. Various embodiments of the presentinvention relate to the use of particular single or combination ofstrains of Lactobacillus in skin probiotics formulations to target skinconditions, such as acne. Streptococcus salivarius is another beneficialbacteria that can be employed, alone or in combination with otherdescribed bacteria as described herein, as being beneficial againstvarious skin conditions, including acne. Such bacteria can beadministered to an individual's skin by way of formulations that mayinclude microcapsules, topical creams, emulsifiers, preservatives, etc.that are selected to be compatible with the bacteria as described, bothduring storage and use of the formulations. Microcapsules can be used tocontain one or more bacterial or probiotic or prebiotic or postbioticmaterials such that they can be delivered to a person's skin withoutmixing or interacting until actually on the skin surface. The rubbing ofsuch formulations on the skin can be employed to break the capsules,releasing the inner core material containing any suspended probiotics.

Early in life, the skin immune system develops abilities to recognizebeneficial microorganisms from pathogenic ones so as to avoid constantimmune responses and inflammation. Certain aspects of the presentinvention relate to seeding of desired bacteria on the skin of a newbornto trigger desired immune responses and/or the maturation of anindividual's immune system.

Another aspect of the present invention is directed to the ability toedit Cutibacterium acnes to remove specific proinflammatory strains andthus prevent or treat acne vulgaris or to modulate host immuneresponses. Thus employing CRISPR systems as described herein, it ispossible to selectively kill or reduce the growth of particularbacteria, thus providing for the desired proliferation of other bacteriaon the skin surface. The use of CRISPR systems may be directed toenhancing the anti-inflammatory effects of Lactobacilli. In variousembodiments of the present invention, CRISPR/Cas9 is used to selectivelydeplete a given bacterial community of a particular harmful strain orspecies, or of particular virulence factors possessed by particularstrains of bacteria.

In certain embodiments of the present invention, antibiotic resistanceof certain bacteria is modulated by employment of CRISPR to insert intothe genome of a bacteria antibacterial sensitivity such that it canselectively be killed, if necessary, after it is employed to triggerdesired immune responses in a newborn or other individual.

CRISPR-Cas systems employ CRISPR RNAs to recognize and destroycomplementary nucleic acids. In various embodiments of the presentinvention, CRISPR-Cas systems are used as programmable antimicrobials toselectively kill bacterial species and strains such that desiredselected targets can be focused on such that virtually any genomiclocation may be a distinct target for CRISPR-based antimicrobials, andthat, in conjunction with an appropriate delivery vehicle, such as thoseemployed by Bikard et al. and Citorik et al., one is able to effectivelydeploy a CRISPR-Cas system as an antimicrobial agent.

Use of CRISPR-Cas provides a generalized and programmable strategy thatcan distinguish between closely related microorganisms and allows forfine control over the composition of a microbial population for use inthe present invention. Thus, the RNA directed immune systems in bacteriaand archaea called CRISPR-Cas systems is employed in various embodimentsof the present invention to selectively and quantitatively removeindividual bacterial strains based on sequence information. Thus, suchgenome targeting using CRISPR-Cas systems allows one to specificallyremove individual microbial species and strains.

In various embodiments, it is desirable to remove—using CRISPR-Cassystems—particular pathogenic bacteria and/or simply the pathogenicportions of such bacteria—while sparing other desired commensalbacteria.

In various embodiments, one of skill in the art will appreciate thatremoval of particular strains of bacteria may be achieved using bothtype I and type II CRISPR-Cas systems, given the distinction betweenthese systems being that type I systems cleave and degrade DNA throughthe action of a 3′-to-5′ exonuclease, whereas type II systems onlycleave DNA. In still other embodiments, multiple guide RNAs can also beused to target several genes at once. The use of effector fusions mayalso expand the variety of genome engineering modalities achievableusing Cas9. For example, a variety of proteins or RNAs may be tetheredto Cas9 or sgRNA to alter transcription states of specific genomic loci,monitor chromatin states, or even rearrange the three-dimensionalorganization of the genome.

CRISPR-Cas can be used on the various identified microbiome constituentsto modify gene expression, including cutting of a gene, repress oractivate a gene, etc. It can be employed to deliver desired regulatorsor any protein to a desired place on a genome of a microbe, thuspermitting one to tailor the attributes of the microbiome of anindividual to promote the health thereof. Because CRISPR-Cas acts beforetranscription occurs, it is able to be employed to target regulatory andother elements on the DNA of microbes that make up the microbiome.

In certain embodiments, C. acnes strains are modified in situ especiallyvia the use of CRISPR systems, and/or are provided via in vitrogenetically modified C. acnes. Various administration methods can beemployed, including the use of Cutibacterium acnes phagemids and/orbacterial cells comprising these phagemids. Skin-resident bacteriaactively engage host immunity through an intact skin barrier, andactivate specific immune cells in a species- and strain-dependentmanner. Cutibacterium acnes (formerly Propionibacterium acnes) is agram-positive rod-shaped aerotolerant bacteria, first isolated from skinin 1897. It belongs to the order Actinomycetales, it is part of thePropionibacteriaceae family and it belongs to the genus Cutibacterium.This genus includes other human skin species such as Cutibacteriumavidum, Cutibacterium ranulosum and Cutibacterium humerusii. C. acnes isone of the most prevalent and abundant bacteria on human skin and can befound both on the skin surface (stratum corneum) and in the hairfollicle.

Embodiments of the present invention encompass unique combinations ofprobiotics, prebiotics, and other skin-beneficial ingredients. Damage tothe skin epithelial barrier is a hallmark of inflammatory skin diseases,such as atopic dermatitis and psoriasis, leading to significantdiscomfort for individuals suffering from these indications. Epithelialbarrier dysfunction can lead to translocation of environmental allergenswhich predispose individuals to the development of atopic and allergicdisease. Additionally, epidermal barrier dysfunction (EBD) is associatedwith use of standard over the counter products for the treatment of acnevulgaris, including xerosis, pruritus, pain, and/or irritant dermatitis.EBD may also lead to an increased propensity to develop rosacea inresponse to environmental triggers. In certain embodiments, aLactobacillus bacteria is employed to generate desired amounts ofmetabolites. For example, in certain embodiments, L. reuteri and/or L.johnsonii are employed, but most preferably, L crispatus is used in abeneficial bacterial composition for topical administration. Certainembodiments may incorporate live bacteria, metabolites of thesebacteria, postbiotics from these bacteria, and/or heat killed bacteria.

One aspect of certain embodiments of the present invention involves thetopical application of Lactobacillus crispatus to an individual's skinto reduce inflammation through production of tryptophan metabolites. Itis believed that such tryptophan metabolites act as AHR agonists. On ofskill in the art will appreciate that embodiments may incorporate livebacteria, metabolites of these bacteria, postbiotics from thesebacteria, and/or heat killed bacteria.

To address the above referenced skin conditions, various embodiments ofthe present invention are directed to both a topical formulation and amethod for using such formulation that includes skin moisturizing agentsin concert with one or more live probiotic bacterial formulations and/orembodiments may incorporate live bacteria, metabolites of thesebacteria, postbiotics from these bacteria, and/or heat killed bacteria.designed to reduce skin irritation, to reduce skin inflammation and tootherwise address certain skin conditions and diseases. In oneparticular embodiment, live bacterial cells of Lactobacillus crispatusare administered to the surface of an individual's skin at a dosage ofat least 10⁸ CFU, preferably in a moisturizing topical formulation, soas to reduce inflammation through the localized production of tryptophanmetabolites. While not bound theory, it is believed that the localizedproduction of tryptophan metabolites by L. crispatus acts as AHRagonists in keratinocytes. Preferably the L. crispatus bacteriaformulation is further combined with a source to maintain the bacteriaon a person's skin for a pre-determined time. In certain embodiments,such a skin formulation includes glycogen. In still other embodiments, aprebiotic for L. crispatus is provided, such prebiotic which may alsoinclude glycogen, and even more preferably it further includes astimulant for the production of ceramide by keratinocytes. The one ormore skin moisturizing agents may include, for example, skin barrierintegrity-enhancing ingredients, such as PEA/MEA bioactive lipids,N-acetyl cysteine, nicotinamide, luteolin, and/or madecassoside. Again,while not bound by theory, bacterial formulations of the presentinvention include at least one bacteria that generates metabolites thatact as AHR agonists. Preferred metabolites comprise tryptophanmetabolites, such as indole-3-aldehyde or indole-3-acetic acid, as wellas those that may reduce inflammation and/or those that inhibit thymicstromal lymphopoietin (TSLP) in keratinocytes in an AHR dependentmanner. It is believed that AhR activation may suppress upregulation ofTSLP expression. In preferred embodiments, L. crispatus is employedunder conditions such that specific metabolites are generated that actas AHR agonists, thereby resulting in the reduction of skininflammation. In preferred embodiments the metabolites generated via theuse of L. crispatus comprise tryptophan metabolites, including at leastone of the following: indole-3-acetic acid, Indole-3-ethanol,Indole-3-pyruvate, indole-3-aldehyde. Thus, aspects of variousembodiments of the present invention include the use of a live bacterialtopical probiotic product that modulates AhR expression through thelocalized production of tryptophan-derived bacterial metabolites.Preferred topical formulations include a combination of live L.crispatus, prebiotic glycogen, and at least onebarrier-enhancing/moisturizing compound.

In certain embodiments the invention is directed to a combination ofmaterials that ultimately are administered to an individual so as toenhance and improve the appearance, health and function (e.g., as abarrier to undesired elements) of the individual's skin surface. Thus,in some embodiments, a particular probiotic bacteria formulation (asdescribed in some detail herein) is combined with select peptides andhuman milk oligosaccharides (HMOs), also known as human milk glycans,e.g. such as can be found in high concentrations in human breast milk,to facilitate the generation of beneficial metabolites by the probioticformulations. In one embodiment, collagen peptides are combined withhyaluronic acid and a probiotic strain, including at least one of aLactobacillus bacteria that utilizes HMO.

In still other embodiments, bacteria are selected for application to anindividual's skin are employed in view of being able to administer thesame to target a particular circadian rhythm. While one focus of thepresent disclosure relates to barrier repair, still other embodimentsare directed to topical lactobacilli and circadian rhythms thereof. Themicrobiome undergoes diurnal variation in composition and function, andthis in turn drives oscillations in host gene expression and functions.Thus, some embodiments of the present invention are directed toaffecting host-microbiome interactions by the appreciation of anindividual's microbiome chronobiology so as to provide a noveltherapeutic approach to treating adverse skin diseases and conditions.

Certain aspects of the present invention are focused on conditionsand/or diseases induced by circadian clock disruption that can bemediated by adjusting an individual's microbiome composition andfunction. Thus, the timing of application of microbiota-basedtherapeutics, such as pre-, pro-, and post-biotics, is employed toadvance the efficacies of treatments as described herein. Anindividual's microbial circadian rhythms due to its diurnal variation istaken into account when administering the type of bacterial formulationsto a person's skin to arrive at preferred therapeutic responses. Thistiming of microbiota-based therapeutics provides a unique method foraddressing skin conditions and diseases in a fashion previouslyunappreciated by those of skill in the art, thus permitting thecorrection of previously unaddressed dysregulation of an individual'scircadian rhythm associated with aging or chronic illnesses, payingattention to such modifications in terms of the diurnally shiftingmicrobiome.

In certain preferred embodiments, L. crispatus is employed andadministered to an individual's skin surface (especially outside thevagina) to address certain skin conditions and diseases as describedherein. Preferably, L. crispatus is combined with a prebiotic,preferably glycogen, in an amount sufficient to sustain the L. crispatusfor at least one hour after application to an individual's skin in amanner that treats a skin condition/disease, such as acne.

In various embodiments, the timing of bacterial and prebiotic andprobiotic applications form aspects of the patentable inventions setforth herein. For example, contacting one's skin with a cleanser, suchas an alcohol, to first remove or kill certain bacteria; followed by aprebiotic; followed by a probiotic; and then a further differentprobiotic bacteria that thrives in the environment generated by thefirst probiotic, is involved in several of the particular embodiments ofthe present invention.

As discussed herein, aryl hydrocarbon receptor (AHR) is aligand-activated receptor expressed in many cell types, includingintestinal epithelial cells. Such agonists include metabolites oftryptophan such as kynurenine (KYN) and/or kynurenic acid (KYNA).Moreover, bacterial metabolites such as SCFAs modulate AHR activity,sometimes not directly as ligands, but nevertheless in a manner tostabilize and facilitate AHR actions. AHR is believed to act as amediator in the communication between the host and a microbiome of thehost, e.g., its gut microbiota. AHR is believed to be activated bymicrobial-specific metabolites of dietary tryptophan.

In certain embodiments, removing or selectively killing certain bacteriaon an individual's skin, followed by purposeful contact of such skinwith a pre-selected bacterial composition, is one aspect of the presentinvention.

A number of inflammatory diseases have manifestations in the skin and/ormucosa, including psoriasis (Ps), atopic dermatitis (AD), contactdermatitis, hidradenitis suppurativa (HS), pyoderma gangrenosum (PG),Sweet's syndrome, mutations in the PSTPIP-1 gene (PAPA syndrome, PAPSHsyndrome and PASH syndrome), Bechet's disease, bullous pemphigold,mucous membrane pemphigold, pemphis vulgaris, cutaneous Crohn's disease,Sjögren syndrome, systemic lupus erythematosus, prurigo nodularis (PN),Pityriasis lichenoides chronica, palmoplantar pustulosis (PPP), pyodermagangrenosum (PG) and erythroderma. Many of these diseases are associatedwith rheumatoid arthritis and inflammatory bowel diseases, suggesting acommon underlying pathophysiology.

Aryl hydrocarbon receptor (AhR) agonists have been used clinically totreat auto-inflammatory diseases including ulcerative colitis, multiplesclerosis, atopic dermatitis and psoriasis. AhR agonists include, forexample, compounds such as indigo and indigo derivatives including,indirubin, meisoindigo, natural-alpha (glycosylated isoindigotin);clinical-stage drugs such as tapinarof, linomide (roquinimex) andlaquinimod; regulatory approved drugs such as itraconazole,ketoconazole, omeprazole, and leflunomide; naturally occurring compoundssuch as FICZ (5,11-dihydro-indolo[3,2-b]carbazole-6-carboxaldehyde,6-Formylindolo[3,2-b]carbazole) and indole derivatives; and polyaromatichydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), coaltar, refined coal tar, and components of tobacco combustion. While therehas been some use of AhR agonists for the treatment of psoriasis andatopic dermatitis, AhR agonists have not been used clinically to treatauto-inflammatory diseases that manifest in the skin.

Certain embodiments of the present invention include the use ofparticular bacterial compositions that include Prevotella to treatcertain skin conditions or diseases, including psoriasis and atopicdermatitis. Preferably, certain strains of Prevotella are employed,alone or in combination with L. crispatus bacteria, and preferably thosebacteria that have been modified using CRISPR systems to remove one ormore virulence factors. Other embodiments include methods of decreasinginflammation in an individual suffering from a skin condition ordisease. Administering to an individual a bacterial compositiondescribed herein, is done to decrease the level of inflammation by theindividual.

Another aspect of certain embodiments of the present invention isdirected to the treatment, reduction and/or prevention of the skincondition acne vulgaris. C. acnes is the most abundant bacteria in thepilosebaceous follicle both in healthy patients and in patients withacne. There are no differences in terms of bacterial load and thus, thefunctionality of C. acnes, rather than just its presence in acnelesions, is important. Certain aspects of the present invention aredirected to the modification of the functionality of C. acnes, e.g. viaemployment of CRISPR systems to affect virulence factors of C. acnes,thus enabling C. acnes to remain present on the skin, but devoid ofundesired functional aspects thereof. As such, certain embodiments ofthe present invention are directed to the modification of C. acnes toremove or reduce the influence of one or more virulence factors of C.acnes. Preferably, such modifications are achieved using a CRISPR systemas herein described. Moreover, the purposeful administration of suchmodified C. acnes to an individual's skin is intended to competitivelycompete with and/or inhibit other bacteria on an individual's skin,including any C. acnes that may remain after a pre-cleansing of anindividual's skin to attempt to reduce the population of un-modifiedbacteria.

C. acnes is the most abundant skin commensal of the human skinmicrobiome and a major component of healthy skin. This exposes a commonmisconception that an individual's microbiome consists of “good bugs andbad bugs” and that all one needs to do is to kill the bad bugs. This isincorrect. Instead, various embodiments of the present invention relateto modifying bacteria that may commonly be deemed to be undesirable or“bad,” and modifying the same so that the beneficial aspects of suchbacteria can be retained while the undesired aspects of such bacteriaare reduced or eliminated. Various embodiments involve the reduction orelimination of virulence factors of otherwise pathogenic bacteria.Competitive advantages can be provided to certain modified bacteria thatpermits them to outcompete other bacteria on a person's skin so as toprovide desired benefits of certain bacteria, including the provisionand generation of desired metabolites for the skin and/or for otherdesired populations of bacteria. Thus, one aspect of the presentinvention relates to preserving the health benefits of C. acnes tohealthy skin, while reducing the harmful and undesired aspects of C.acnes in its propagation of acne vulgaris. One particular virulencefactor to be targeted in certain embodiments of the present invention isthe virulence factor denoted the tight adherence (tad) locus. C. acnesis known to outcompete many other bacterial species, which may be due toits genome stability or to defense strategies that disallow geneticchanges. C. acnes clade II strains contain full CRISPR/Cas type I-Esystems with spacers matching phages. In comparison, C. acnes clade Ihas lost big parts of such systems over time, indicating that theyrepresent a more recent evolutionary lineage compared to clade IIstrains. Phages within the skin modulate skin microbiome composition byinfecting either a narrow range of strains or broadly within apopulation. It is believed that a healthy skin microbiome harbors morephages than an unhealthy one and C. acnes harbors multiple temperate andlytic phages. Thus, various embodiments of the present invention includethe modification of either C. acnes or bacteriophages thereof to providea more healthy skin bacterial population.

A link exists between the presence of intestinal dysbiosis and theimbalance of skin homeostasis, with the intestinal microbiota playing arole in the pathogenesis of several inflammatory skin diseases. Whilethe human intestine houses 70% of the body's immune cells, the skin isalso rich in immune cells and is densely colonized by bacteria and thereis a bidirectionality between the intestinal microbiota and skinhomeostasis. Thus, while the intestinal microbiota has a major influenceone's immune system, the skin microbiota is important in maintaining anadequate immune homeostasis of an individual's skin.

The severity of acne is at least partially due to an inflammatoryresponse related to the presence of C. acnes, as well as an excess ofsebum, hormonal influences, immune responses influenced by theintestinal microbiota, and/or an individual's genetic predisposition.The present inventors contend that the presence of C. acnes withvirulence factors in acne lesions is one reason for the differentfrequency of appearance of different phylotypes in acne lesions. C.acnes has a constant core region and a variable region in its DNAsequence, and thus the variable region is believed to dictate theobserved variability with respect to its commensal and pathogenicphenotypes and its consequential influence on the development of acne.Among the important virulence factors of C. acnes are fiveChristie-Atkins-Munch-Petersen (CAMP) factors identified in the genomeof all C. acnes. These are secreted toxins that cause the formation ofpores in the membranes and are potentially cytotoxic to keratinocytesand macrophages, causing inflammation of the skin. Porphyrins aremetabolites that generate reactive oxygen species (ROS) and can lead toinflammation in keratinocytes. C. acnes phylotypes related to acneproduce more porphyrins. The generation of ROS activates inflammatoryprocesses, such as lipid peroxidation of sebum lipids. Because virulentstrains of C. acnes that are associated with acne produce porphyrins, itis believed that Vitamin B12 supplementation may be counterproductive inacne patients as porphyrins are involved with Vitamin B12. In variousembodiments of the present invention, an individual does not takeVitamin B12 supplementation in order to reduce the incidence of acnevulgaris.

There are two variants of hyaluronate lyase found in C. acnes: HYL-IB/IIand HYL-IA. Hyaluronate lyase is an enzyme capable of degradinghyaluronic acid and other glycosaminoglycans from the extracellularmatrix of skin cells, contributing to inflammation in acne lesions.HYL-IA performs an incomplete degradation of hyaluronic acid, whileHYL-IB/II causes a complete degradation thereof. Various embodiments ofthe present invention involve the reduction or elimination ofhyaluronate lyase in C. acnes, preferably HYL-IA.

Another virulence factor of C. acnes includes lipases. Lipases areenzymes that metabolize lipids of the sebum, generating FFAs. It hasbeen shown that C. acnes encodes 12 lipases, including glycerol-esterhydrolase A (GehA) and B (GehB), which are present in strains of C.acnes. C. acnes lipases are important in the context of acne becausethey are involved in the production of FFAs that promotes inflammation.Various embodiments of the present invention involve the reduction orelimination of lipases, including one or more of glycerol-esterhydrolase A (GehA) and B (GehB).

Other virulence factors of C. acnes associated with acne are the enzymesneuraminidase, PUFA isomerase, gliycosidase, sortase F, and RoxP,heat-shock proteins and dermatan-sulfate-binding adhesins. Variousembodiments of the present invention involve the reduction orelimination of these virulence factors, preferably employing CRISPRsystems to do so.

Strains that belong to phylotype II of C. acnes are most associated withhealthy skin while phylotype I strains are most associated with acnelesions. The latter usually present deletions in regions of CRISPR/Cassequences, which implies a greater capacity to acquire virulence factorsthrough horizontal transmission.

The ability to form biofilms, i.e. a structured colony of bacterialcells embedded in a polymer matrix that is manufactured by the bacteriaand adhered to a surface, is a particularly important virulence factorof C. acnes. Biofilms formed by C. acnes are significantly higher inacne lesions than in the skin of healthy controls. Formation of biofilmsby C. acnes is at least partially due to the presence of genes involvedin such formation, such as glycosyltransferases and uridine diphosphateN-acetylglucosamine 2-epimerase. Thus, in certain embodiments thepresent invention is directed to the reduction or elimination of thesegenes, preferably employing CRISPR systems to do so. As the presence ofbiofilms is associated with a greater resistance to antibiotics due tothe presence of an exopolysaccharide matrix that prevents or hinders theinteraction of an antibiotic to its molecular target, the reduction orelimination of biofilms forms one aspect of the present invention as itrelates to the treatment of acne vulgaris.

Certain embodiments are directed to treating an individual sufferingfrom acne vulgaris, by using a clustered regularly interspaced shortpalindromic repeats (CRISPR) CRISPR associated protein (Cas) toselectively kill or retard the growth of a pathogenic bacteria withinthe individual, the pathogenic bacteria selected from the groupconsisting of Staphylococcus aureus; Pseudomonas aeruginosa; Klebsiella;Streptoccocus; Salmonella; Shigella; Mycobacterium tuberculosis;Enterococcus; E coli; Clostridium; Neisseria gonorrhoeae; Acinetobacterbaumannii; and Campylobacter, and thereafter enhance the growth of abeneficial bacteria in the individual selected from the group consistingof C. acnes, Akkermansia, Bacteroides, Bifidobacterium, Fusobacterium,Coprococcus, Lactobacillus, Propionibacterium, Ruminococcus,Veillonella, Prevotella, and Streptococcus bacteria. Still otherembodiment are directed to treating acne vulgaris by administering tothe skin of an individual at least two probiotic strains in aconcentration of at least 1×10⁸ via AFU, with the at least two probioticstrains being either live or heat-killed and selected from the groupconsisting of Cutibacterium, Staphylococcus, Corynebacterium,Micrococcus spp., Actinobacteria, Proteobacteria, and Firmicutes. Otherembodiments are directed to treating acne vulgaris by administering atleast one probiotic strain to an individual's skin that does notupregulate innate immune response genes selected from the groupconsisting of CXCL1, CXCL3, CXCL8, CXCL10, wherein the strains areeither live or heat-killed. Yet other embodiments are directed totreating acne vulgaris by administering to an individual's skin at leastone probiotic strain that does not upregulate innate immune responsegenes selected from the group consisting of IL10RA, PTGS2, F2RL1,TRIM29, TRAF4, LGALS3, CD55, TRIMS, CASP4, IFNGR1, ADA, NOD1, NOS2, andAPP.

It should be understood that still other embodiments of the presentinvention include the employment of a variety of postbiotic formulationsof lactobacilli, e.g. metabolites or lysates, and thus, is not limitedto the use of live bacteria formulations or methods. Some embodimentsinvolve the use of strains that are heat-killed. Indeed, in preferredcompositions and formulations, in addition to the bacterial aspects andmetabolite inclusions of described products, there are additionalingredients, such as skin moisturizing agents as described herein and inreferences incorporated herein by reference, with several compositionsincluding heat killed bacteria.

Still other embodiments are directed to compositions, systems andmethods of improving the health of the microbiome of an individual'sskin and treating an individual suffering from acne vulgaris byadministering topically a composition that includes live bacteriaselected from the group consisting of L. reuteri, L. johnsonii, L.crispatus, C. acnes, and Nitrosomonas eutropha, that have been modifiedby using a using a clustered regularly interspaced short palindromicrepeats CRISPR) CRISPR associated protein (Cas) system or a CRISPR fromPrevotella and Francisella 1(Cpf1) system to reduce the production of avirulence factor of the bacteria.

One will appreciate that this Summary of the Invention is not intendedto be all encompassing and that the scope of the invention nor itsvarious embodiments, let alone the most important ones, are necessarilyencompassed by the above description. One of skill in the art willappreciate that the entire disclosure, as well as the incorporatedreferences, figures, etc. will provide a basis for the scope of thepresent invention as it may be claimed now and in future applications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a picture of Faecalibacterium prausnitzii.

FIG. 2 is a picture of Akkermansia muciniphila.

FIG. 3 is a picture of Roseburia.

FIG. 4 is a picture of Clostridium.

FIG. 5 is a picture of Veillonella.

FIG. 6 is a picture of Prevotella.

FIG. 7 is a picture of Propionibacterium.

FIG. 8 is a picture of Pseudomonas aeruginosa.

FIG. 9 is a picture of Klebsiella

FIG. 10 is a picture of Shigella.

FIG. 11 is a picture of Acinetobacter baumannii.

FIG. 12 shows where various bacteria reside on the human skin, whichvaries by region of the body and that depends upon whether the skin siteis oily, moist or dry.

FIG. 13 is a picture of L. crispatus.

FIG. 14 is a picture of Lactobacillus reteri.

FIG. 15 is a picture of Lactobacillus johnsonii

FIG. 16 is a picture of Nitrosomonas eutropha.

FIG. 17 is a picture of Cutibacterium acnes.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

The present invention stands in contract to accepted methods of dealingwith skin and bacteria issues (which largely solely involve killingbacteria, etc.—such as described in Kimberly Clark's U.S. Pat. No.8,110,215 to Koenig, et al.) In contrast, various embodiments of thepresent invention are directed to modification of various bacteria on aperson's skin so as to reduce the pathogenicity thereof and to rely uponcompetitive inhibition of such modified bacteria on the skin to reducethe presence of pathogenic bacteria on an individual's skin.

As set forth above, in particular embodiments of the present invention,contacting the skin of a newborn is performed to address the propertriggering of the newborn's immune system development. Thus, certainaspects of the present invention are directed to a method for alteringthe microbiome of an individual's skin by administering to a region ofthe skin of a newborn within the first 6 hours of the newborn's birth aparticular bacterial formulation. Such a bacterial formulation may be alotion, ointment or gel adapted to be rubbed onto the newborn's skin.The bacteria included in the bacterial formulation may vary to addressparticular concerns or diseases. For example, the bacterial formulationmay include bacteria selected from the group consisting of Nitrosomonaseutropha and Propionibacterium. More particularly, the equilibrium of abacterial population of the region of the skin of the individual ismodified to increase the number of Propionibacterium bacteria and todecrease the number of Staphylococcus bacteria on the individual's skinin such region. In other embodiments, the bacterial formulation includesthe bacteria Staphylococcus aureus that has been modified by employing aCRISPR-Cas or Cpf1 system to interfere with S. aureus virulenceregulation involving the Agr quorum-sensing signaling molecule. Inseveral embodiments, the bacterial formulation comprises a bacteria thathas a tropism specific for the human species. In others, the bacterialformulation comprises at least two of the bacteria selected from thegroup consisting of: Prevotella; Lactobacillus johnsonii; Bacteroidesfragilis, Lactobacillus ruminis and L. infantitis. In certainembodiments the bacteria is an ammonia oxidizing bacteria. In otherembodiments, the region of the skin to which the bacterial formulationis applied is the scalp. In various embodiments, rather than using awild-type bacteria, the bacteria employed is one that has been modifiedby CRISPR-Cas or CRISPR-Cpf1 to delete a functional virulence factorfrom the bacteria. In particular embodiments, the method includesadministering to the skin a bacteria that produces tomatidine. Someembodiments involve the application of an effective amount of atomatidine containing formulation to an individual's skin as tomatidineis a steroidal alkaloid from solanaceous plants that possesses potentantibacterial activity against S. aureus. In others, the bacteriaproduces p53. Thus, in some embodiments, the method involves use ofbacteria wherein a CRISPR-Cas or CRISPR-Cpf1 system is employed toinsert a gene for the production of tomatidine and/or p53 into at leastone of the bacteria in the bacterial formulation. In others, aCRISPR-Cas or CRISPR-Cpf1 system is employed to insert one or more genesinto the bacteria comprising the bacterial formulation to facilitate theoxidizing of ammonia by the bacteria. To further enhance the ability ofdesired bacteria to be maintained on the skin of an individual, certainmethods further comprise administering to the individual's skin aprebiotic that comprises a nutrient source for the bacteria that isassimilated by the bacteria, and preferably one that is not digestibleby the individual. In particular embodiments, the method furtherincludes administering to the skin an extract derived from a helminthselected from the group consisting of Capillaria hepatica, Dicrocoeliumdendriticum, Ascaris lumbricoides, Enterobius vermicularis, Trichuristrichiura, Ancylostoma duodenale, Necator americanus, Strongyloidesstercoralis, Haemonchus contortus, and Trichinella spiralis. In stillothers, the bacterial formulation includes at least one arabinogalactan.Yet others include at least one of the following: L. infantitis, and L.johnsonii. In a particular embodiment, the bacterial formulationincludes at least one bacteria modified via a CRISPR-Cas system toexpress a gene encoding interferon regulatory factor 4.

As for lotions of the present invention, in preferred embodiments, thereis an objective to limit if not preclude the use of phthalates, whichare extremely toxic and are believed to also be human carcinogens. Thus,in preferred embodiments of the present invention, such lotions do notemploy such toxic agents, and in particular agents toxic to bacterialspecies for which the inventors suggest be used, e.g. those modified toreduce pathogenicity, virulence factors, etc, so as to establish apopulation of such modified bacteria on a person's skin, and in such amanner, reduce the incidence of skin infections and diseases. Thus,lotions, creams, gels, etc. that include such toxic agents, includingbut not limited to phthalates, are not employed, but rather, lotionsthat provide an environment for the bacteria as set forth herein tosurvive and to thus be available to provide benefits to the skin ofindividuals to which they are applied, are particularly preferred.

Healthy, normal skin exhibits a slightly acidic pH in the range of4.2-5.6, which aids in the prevention of pathogenic bacterialcolonization, regulation of enzyme activity, and maintenance of amoisture-rich environment; however, after the age of 70, the pH of skinrises significantly, stimulating protease activity. Thus, one objectiveof several embodiments of the present invention is directed to loweringthe pH of the skin of an individual, especially those at about the ageof 70, so as to encourage a skin environment conducive to theproliferation of one or more bacteria that have been modified to promoteskin health and to reduce the ability of undesired bacteria fromcolonizing the skin of the person. Probiotic metabolism frequentlyproduces acidic molecules, lowering the pH of the surroundingenvironments seen with Lactobacilli producing free fatty acids (FFAs)and conjugated linoleic acid (CLA) during the fermentation process.Thus, the use of probiotics is employed to restore the normal skin pHand consequently return protease activity levels closer to those seen inyoung, healthy skin.

The main microbes that reside on human skin can be divided into fourphyla: Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes.Staphylococcus spp. and Corynebacterium spp. are the dominant bacteriaat the genus level.

Significantly fewer Corynebacterium spp. have been observed in cachexiapatients compared to healthy subjects. These results suggest that thepresence of cancer and cachexia alters human skin bacterial communities.Understanding the changes in microbiota during cancer cachexia may leadto new insights into the syndrome.

Competitive inhibition is relied upon in various embodiments to advancethe repopulation of skin with beneficial microbes. In one embodiment,repopulating an individual's skin with beneficial bacteria, preferablyin balanced percentages and having preferred species provided, can beused in conjunction with an antimicrobial composition. Preferably, anantimicrobial is first administered to suppress or eradicate theresident populations of bacteria on a person's skin, including anyabnormal organisms or pathogenic bacteria, then the normal flora isrepopulated by the administration of at least one of the modifiedbacteria as described herein, including those modified using CRISPR-Casand/or Cpf1 systems to delete certain portions of genes or to addcertain genes to facilitate the colonization of a person's skin withbeneficial bacteria that maintain the general health of a person's skin.

It is preferred that the antimicrobial treatment is complete before theadministration of modified bacteria that are desirable to maintain skinhealth, including modified bacteria of the following: Firmicutes (mainlyStreptococcus and Staphylococcus) and Actinobacteria (mainlyCorynebacterium and Propionibacterium). By employing such modifiedbacteria, one is able to establish and maintain the reduction if notpreclusion of various skin diseases, including skin cancer. Oneobjective of the present invention is to provide a method and systemthat, by using health promoting strains from the microbiome in topicalprobiotics, it is possible to treat and to further reduce the risk ofskin cancer.

Repair of tissue wounds is a fundamental process to re-establish tissueintegrity and regular function. Infection is a major factor that hinderswound healing. Multicellular organisms have evolved an arsenal ofhost-defense molecules, including antimicrobial peptides (AMPs), aimedat controlling microbial proliferation and at modulating the host'simmune response to a variety of biological or physical insults. Certainembodiments of the present invention are directed to the use of AMPs asendogenous mediators of wound healing. Thus, one aspect of severalembodiments of the present invention is directed to geneticallymanipulating bacterial species native to the skin. Staphylococcusepidermidis, which is found in abundance on human skin, can cause immunetolerance in some—but in others, inflammation and activation of T cellsagainst the bacteria. The present inventors submit that the immunesystem may set up tolerance to commensal bacteria only early in life,during a time where there is an influx of regulatory T cells unique tothe skin, e.g. during the first week after birth. This colonization ofthe skin by regulatory T cells—immune cells that dampen the responses ofeffector T cells—is believed to be required for tolerance to S.epidermidis. There is an abrupt wave of regulatory T cell infiltrationinto neonatal skin that occurs at a defined period and this windowdictates the achievement of commensal-specific tolerance.

One aspect of the present invention is directed to the introduction oftolerance to commensal bacteria during the time the developmental windowis still open, thus providing the individual with life-long protectionfrom a variety of diseases. Still other embodiments, however, aredirected to introducing tolerance following the closing of thedevelopmental window, e.g. after the first week after birth, so thatindividuals can purposefully be induced to have commensal-specifictolerance as an adult. Understanding which microbes cause infection andwhich are tolerated and the critical time frames where the immune statusis set is one aspect of the present invention.

Skin bacterial communities are influenced by ethnicity, lifestyle and/orgeographic location. Skin bacterial communities that are particularlyemployed in the modifications as set forth herein include: Firmicutes,Proteobacteria and Actinobacteria); Firmicutes (mainly Streptococcus andStaphylococcus) and Actinobacteria (mainly Corynebacterium andPropionibacterium), while still other preferred bacteria include L.acidophilus NCFM, L. salivarius Ls-33, Bifidobacterium lactis 420, L.acidophilus La-14 and Propionibacterium jensenii P 63.

In various embodiments, cosmetics are provided that provide for a mediumfavorable for maintaining a desired physico-chemical balance of the skinwithout favoring the development of pathogenic microorganisms. Toachieve this objective, certain oligosaccharides that are metabolized byseveral beneficial strains of the skin microflora, such as Micrococcuskristinae, Micrococcus sedentarius, Staphylococcus capitis,Corynebacterium xerosis and Lactobacillus pentosus, are employed informulations, in conjunction with one or more of the modified bacteriaas described herein.

Pathogenic strains such as Staphylococcus aureus, Gardnerella vaginalisand Propionibacterium acnes do not typically metabolize, or veryslightly metabolize, these oligosaccharides. In certain embodiments,these sugar sources are provided in amounts and in association withbeneficial bacteria, whether they be those modified as described herein,or those that are naturally non-pathogenic in nature, so as to achievethe colonization of the skin in a fashion to provide the health benefitssought. In particular embodiments, oligosaccharides are employed informulations for the skin that include one or more of Lactobacilluspentosus, Micrococcus kristinae, Gardnerella vaginalis,Propionibacterium avidum and Propionibacterium granulosum. As statedherein above, it is often beneficial to further acidify the culturemedium, and this can be achieved, for example, by employing Lactobacillito produce in particular lactic acid to achieve pH reducing effects.

In certain embodiments, the present invention is directed to cosmeticcompositions having at least one oligosaccharide chosen from the groupconsisting of gluco-oligosaccharides, fructo-oligosaccharides, andgalacto-oligosaccharides and mixtures thereof. In addition to theoligosaccharide constituent, the cosmetic compositions of particularembodiments of the invention may contain other ingredients, but cautionis warranted as one objective is to avoid incorporating ingredientswhose properties would interfere with the development of the beneficialskin microflora and the preservation of acidic conditions. Thus, it isadvisable to avoid incorporating bactericidal ingredients in proportionswhich would annihilate the endogenous microflora, or ingredients whichconfer a pronounced basic character on the composition. For example, inpreferred embodiments, reduction if not elimination of ionicsurface-active agents, such as sodium lauryl sulfate, is advisable, aswell as other well-known agents having bactericidal properties. Instead,use of a non-ionic surface-active agent such as an alkyl glucoside or adialkyl ester may be employed in various embodiments. Preferably,cosmetic compositions of the invention contain an acidic buffer whichadjusts the pH of the composition to about pH 4 to 7 range, preferablyabout 5 to 6.5 pH. At such range, especially on the lower side,mutualistic flora such as Staphylococci, Micrococci, Corynebacterium andPropionibacteria preferably grow but not transient bacteria such as Gramnegative bacteria like Escherichia and Pseudomonas or Gram positive onessuch as Staphylococcus aureus or Candida albicans.

One aspect of the present invention relates to cosmetic products thatinclude skin probiotics having viable organisms purposefully included,especially those genetically designed (as by CRISPR systems) so as toconfer health benefits to the skin without the dangers of bacterialinfections and inflammation. Indeed, certain other aspects of variousembodiments are directed to the reduction of body odor by use of aprobiotic skin formulation that can be provided to consumers as alotion, spray, roll-on etc. Thus, stimulating the growth of certainbacteria and microbes, while deterring the growth of others, to arriveat an acceptable odor prevention formulation, is one of the generalobjectives of various embodiments.

Certain other embodiments are directed to the rebalancing of the skinmicrobiota using antimicrobials with selective action. For example, incertain embodiments a balance of species and characteristics is soughtto provide skin formulations that maintain a well-balanced bacterialflora, and especially one that includes one or more of the modifiedbacteria as described herein. Thus, one particular aspect of variousembodiments is directed to the provision of embodiments targeted toreduce undesired body odor (and in various embodiments, activelyprovides microbes that generate desired odors and reduces the effects ofmalodors by other bacteria) which can be gender specific.

Thus, in certain embodiments, a system and method is provided thatoffers the interactions between bacteria and precursors of thiols—anorganosulfur compound responsible for some of the more pungent qualitiesof onion, garlic and human sweat. In human adults, smell associated withsweat originates from apocrine glands located in the armpit, and theodor results from the degradation of the excretion of these glands bybacteria in the armpit.

Particular embodiments are directed to anti-deodorants used specificallyfor under a person's arm. In various formulations of the presentinvention, the use of bacteria able to generate lactic acid to serve asa moisturizing factor, still others that produce hyaluronic acid toimprove skin hydration and elasticity, and that include sphingomyelinaseto generate ceramide to enhance skin barrier function, are preferredcompositions. As one of skill in the art will appreciate, while variousembodiments of the present invention are directed to cosmetics, othersare admittedly directed to formulations having claims for effects thatinclude skin protection, and modification of cellular structure orfunction and thus, may be considered a drug under the FD&C Act. Oneaspect of the present invention is directed to restoring homeostasis totreat certain skin diseases by remedying the dysbiosis in the skinhabitat by establishing a desired colony of various diverse bacteria,especially those modified as described herein to establish and maintaina healthy skin condition on an individual's skin.

The antiperspirant market is currently dominated by topically appliedproducts based on aluminum or zirconium salts which are intended toprevent, or at least control, localized perspiration at the skinsurface, particularly on the underarm. Deodorants are formulations thatare designed either to mask malodor or to prevent or hinder itsformation. The latter method usually comprises reducing and/orcontrolling the re-growth of the local micro-organism populations, ortargeting preferentially those bacteria such as a sub-class ofCorynebacteria which contribute disproportionately to axillary odorgeneration, or interrupting the pathways by which malodors are formedfrom secretions. Aluminum or zirconium salts provide deodorancy benefitseven at a level below the commonly accepted threshold for significantantiperspirancy to be observed.

A principal disadvantage of many antiperspirants is that they containone or more commonly employed ingredients which are perceptiblyunfriendly to human skin in those areas of the body to which theformulations are normally applied. Such ingredients are perceived toexhibit an adverse effect, in particular an irritant effect, on a user'sskin following application of the antiperspirant salt-containingformulation. In one embodiment of the present invention, bacteriaspecies are employed that have been modified via CRISPR-Cas systems toreduced malodor without the employment of aluminum or zirconium salts.Such modified bacteria suppress malodor and counteract or suppress sweatmalodor. Even more preferred bacteria have been modified to expresscompounds of a pleasant and desirable scent. Such bacteria can thusprovide amounts of a perfume scent that is pleasant to a person and thatcan at least partially mask the unpleasant body odor smells produced bya person. Splicing in such “perfume” genes into bacteria using theCRISPR-Cas system is one way to accomplish this objective. Use of suchbacteria on a person's skin, and in particular under armpits where theparticular type of bacteria is selected to grow and out-complete othermicrobes in such a moister environment (as compared to elbows, etc.) canbe used to enhance the desired smells of one's body while limiting theamount of traditional antiperspirants and deodorants conventionallyemployed. Still other embodiments include the use of bacteria thatutilize as their food source the very bacteria that produce malodors. Insuch a fashion the desired bacteria feed off of the products produced byundesired bacteria on a person's skin, and in particular under anindividual's arm, so that undesired body odor is reduced and without theuse of traditional chemicals and compounds as previously discussed.

To further comply with written description and enablement requirements,the following patents and patent publications are also incorporatedherein by this reference in their entireties: are the following: U.S.Pat. No. 8,815,538 to Lanzalaco, et al.; 20150374607 to Lanzalaco, etal.; 20150361436 to Hitchcock et al.; 20150353901 to Liu et al.; U.S.Pat. No. 5,518,733 to Lamothe, et al.; 20150259728 to Cutliffe et al.U.S. Pat. No. 8,685,389 to Baur; 20140065209 to Putaala et al.; U.S.Pat. No. 8,481,299 to Gueniche; WO 2011029701 to Banowski; 20150071957to Kelly; 20150202136 to Lanzalaco; 20150017227 to Kim; U.S. Pat. No.7,820,420 to Whitlock; 20150202136 to Lanzalaco et al.; U.S. Pat. No.5,518,733 to Lamothe, et al.; U.S. Pat. No. 8,815,538 to Lanzalaco et.al; U.S. Pat. No. 8,951,775 to Castiel; WO 2006/07922; U.S. Pat. No.9,234,204 to Qvit-Raz et al.; U.S. Pat. No. 8,758,764 to Masignani, etal.; U.S. Pat. No. 9,028,841 to Henn et al.; 20160008412 to Putaala etal., 20150064138 to Lu; 20150017227 to Kim; United States PatentApplication No. 20160314281 to Apte; 20160151427 to Whitlock et al.;20140044677 to Raz et al.; 20160168594 to Zhang et al. U.S. Pat. Nos.7,267,975; 9,288,981; United States Patent Application No. 20160122806;9234204 to Noga Qvit-Raz; US20120301452; 20160271189 to Cutcliffe; USPat. Applic. No. 2008242543; 20160040216 to Wilder; and United StatesPatent Application No. 20160089315 to Kleinberg, et al. and 20070148136to Whitlock et al.; 20200190494 to Hou, et. al.; and 2020/0199555 toZhang; U.S. Pat. No. 9,585,920; al., 20190059314 to Aharoni; 20200009268to Scholz and 20200009185 to Shin; US Pat. Publication No. 20190388471to June; 20190000815 to Melin; 20180258100 to Gregory; 20170027914 toQi; 20170079947 to Richards; 20140296139 to Cohen et al.; 20160175327 toAdams et. al.; 20100081681, 20130310416, and 20120283269 toBlagosklonny; U.S. Patent Publication Nos. 20140030332 to Baron, et al.,20070123448 to Kaplan et al.; 20160000841 to Yamamoto, et al.;20160095316 to Goodman et al.; 20160158294 to Von Maltzahn; 20140294915to Kovarik; U.S. Pat. No. 8,034,601 to Boileau et al.; 20130225440 toFreidman, et al., 20150071957 to Kelly et al., 20160151428 to Bryann etal.; 20160199424 to Berry et al.; 20160069921 to Holmes, et al.;20160000754 to Stamets; U.S. Pat. No. 9,044,420 to Dubensky, Jr, et al.;20160120915 to Blaser et. al.; 2014/0349405 to Sontheimer; 2014/0377278to Elinav; 2014/0045744 to Gordon; 2013/0259834 to Klaenhammer;2013/0157876 to Lynch; 2012/0276143 to O'Mahony; 2015/0064138 to Lu;2009/0205083 to Gupta et al.; 201/50132263 to Liu; and 2014/0068797 toDoudna; 2014/0255351 to Berstad et al.; 2015/0086581 to Li;PCT/US2014/036849 and WO 2013026000 to Bryann; U.S. Pat. Publication No.2015/0190435 to Henn; 2012/0142548 to Corsi et al.; U.S. Pat. Nos.6,287,610, 6,569,474, U.S. 2002/0009520, U.S. 2003/0206995, U.S.2007/0054008; and U.S. Pat. No. 8,349,313 to Smith; U.S. Pat. No.9,011,834 to McKenzie; 20150004130 to Faber et. al, 20160206666 to Falb;20160206668 to Kort et al; and WO2015069682A2 to Asesvelt, et. al.;20160199424 to Berry et al.; 20130326645 to Cost et al.; 2012/0276149 toLittman; U.S. Pat. No. 9,314,489 to Kelly et al.; U.S. Pat. PublicationNo. 2016/0024510 to Bikard et al.; U.S. Pat. Publication No.2018/0015131 to Gajewski et al.; U.S. Pat. Publication No. 2018/0371405to Barrangous et al. and U.S. Pat. Publication No. 2018/0140698 to Clubeet al.; 20160199424 to Berry et al.; and 20130326645 to Cost et al.;20190315642 to Parsley; 20170246269 to Hajishenfallis; 201302236488 toDashper; 20030083287 to Burgess; 20170042860 to Kashyap; 20150045546 toSiksnys; U.S. Pat. No. 10,730,826 and 20200148642 to Konradi et. al.;202110321756 to McLaughlin, et. al.; 20210308028 to Yang et. al.; U.S.Pat. No. 11,083,760 to Han; 20180110795 to Frias-Lopez; 202110169954 toBalani et. al.; U.S. Pat. No. 4,720,486 to Spilburg; and 20070207955 toTanihara; U.S. Pat. No. 11,473,093 to Levenau, et. al.; 20220339208 toAbel, et. al.; 20220331375 to Kovarik; 20210361560 to Krueger, et. al.;WO2019067621 to Bardales, et. al.; 20220331374 to Richter, et. al.;20220339208 to Abel, et. al.; 20220023259 to Davidson et. al.;20180235987 to Von Maltzahn, et. al. and WO 2022/187274 to Ratti.

Skin is composed of a variety of niches, including regions with a broadrange of pH, temperature, moisture, and sebum content. Furthermore, skinstructures such as hair follicles, sebaceous, eccrine, and apocrineglands comprise subhabitats that may be associated with their own uniquemicrobiota. Microorganisms that colonize the skin include Staphylococcusepidermidis and other coagulase negative staphylococci. Othermicroorganisms that are generally regarded as skin colonizers includecoryneforms of the phylum Actinobacteria (the genera Corynebacterium,Propionibacterium and Brevibacterium) and the genus Micrococcus.Modification of such bacteria via CRISPR-Cas and/or Cpf1 systems toenhance positive and beneficial aspects of such bacteria is one aspectof various embodiments of the present invention.

One aspect of certain embodiments is directed to the topicaladministration of probiotic bacteria, and/or soluble metabolites ofprobiotic bacteria and/or a cell lysates of probiotic bacteria that canimprove Tight Junction (TJ) function in the epithelium. The probioticbacteria is preferably provided in the form of a soluble metabolite witha formulation in a cream, lotion, spray, solution, gel, ointment,bioadhesive or suspension, or strip, especially one having encapsulatedformulations inside. The formulations described herein are markedlydifferent from natural counterparts due to their modification, e.g. viaCRISPR-Cas systems as described herein. While some embodiments aredirected to a product, others are directed to a method, and thus, thelater are not all limited to the products described herein.

Thus, in one embodiment the invention relates to a cosmetic method fortreating a non allergic irritant contact dermatitis of an individual inneed thereof by administering to an individual an active agentcomprising an effective amount of at least one a probiotic microorganismselected from the genus Lactobacillus sp. and/or Bifidobacterium sp.,preferably Lactobacillus johnsonii to limit skin irritation, and inparticular, a skin infection caused by methycilin-sensitiveStaphylococcus aureus. Administration is preferably by topicalapplication of a pharmaceutical composition comprising at least about1.5% by weight of the bacterium.

The skin is a natural barrier to the penetration of foreign substances.As the skin barrier is compromised, the skin is subject to inflammatoryevents from percutaneous absorption of irritants through the stratumcorneum. Skin barrier function can be compromised by environmentalirritants, mechanical abrasion, continuous tissue load pressure,exposure to body fluids and waste such as proteases, ureases andlipases, and especially those that cause an alkaline pH, and exposure tochemicals. Personal care products are an integral part of people'sroutines and habits, and thus, one aspect of the present invention is toprovide skin probiotics to contribute to the promotion of human health.

Staphylococcus epidermidis and Staphylococcus aureus make up about 5% ofskin bacteria, with the diverse culture on the skin also includingbacteria primarily from four phyla: Actinobacteria, Firmicutes,Proteobacteria, and Bacteroidetes. It is known that females generallyhave more Staphylococcus living in their skin microbiomes (usuallyStaphylococcus epidermidis) and that men have more Corynebacteriumliving in their skin microbiomes.

Properly adjusted, the skin microflora can aid in immunity developmentand maintenance. In certain embodiments, CRISPR-Cas or Cpf1 systems areemployed to modify Pseudomonas aeruginosa, typically a mutualisticbacterium but one that can turn into a pathogen and cause disease. Usingthe referenced CRISPR systems, virulence factors are compromised orexcised in a manner that makes such bacterium much safer to employ, andreducing substantially its pathogenic capabilities, such that populationof a person's skin with such modified bacteria causes conditions ofcompetitive inhibition of other pathogenic wild type bacteria of thesame species. The use of such modified bacteria is therefore useful inpreventing wild type strains form gaining entry into the blood systemwhere it is known to cause infections in bone, joint, gastrointestinal,and respiratory systems, as well as dermatitis. Moreover, whilevirulence factors are excised or reduced by modification, other aspectsof the bacteria re maintained, such as its production of antimicrobialsubstances such as pseudomonic acid, such that infections caused bystaphylococcal and streptococcal are reduced, as well as the growth offungus species such as Candida krusei, Candida albicans, Torulopsisglabrata, Saccharomyces cerevisiae and Aspergillus fumigates, andimportantly, the use of such modified bacteria can inhibit the growth ofHelicobacter pylori. Other bacteria that may be modified to reduce ifnot eliminate various compounds from being produced by bacteria thatordinarily are known to generate undesired orders includePropionibacteria, which can turn amino acids into propionic acid,Staphylococcus epidermidis, which breaks human sweat into isovalericacid (3-methyl butanoic acid), and Bacillus subtilis, which createsmalodorous compounds that lead to foot odor.

In certain embodiments, one aspect of the present invention is directedto the treatment of acne by using probiotic treatments that includeeffective amounts of Staphylococcus epidermidis and/or Lactobacillusplantarum to inhibit P. acnes growth, which are believed to producesuccinic acid, shown to inhibit P. acnes growth. CRISPR-Cas and/or Cpf1systems are used to modify such bacteria in a manner that reduces theoccurrence of acne, such as by altering the expression of genes so thatthe amount of succinic acid on a person's skin is increased.

Still other aspects of certain embodiments are directed to treatingindividuals with atopic dermatitis, a disease that is associated withlow bacterial diversity due to colonization by S. aureus, with suchtreatment including the purposeful application of a formulation thatcontains effective amounts of S. epidermidis to inhibit S. aureusgrowth. Preferably the skin bacteria population should demonstrate highlevels of Bacteroides and low levels of Firmicutes. Thus, application ofprobiotics to a person's skin to achieve such a desired ratio ofbacterial species is one aspect of various embodiments of the presentinvention, with preferred embodiments including the use of CRISPR-Cassystems to modify selected bacteria in a manner that enhances theirabilities to reside on a person's skin.

Yet another aspect of certain embodiments is directed to addressing thepopulation of Psoriasis vulgaris on a person's skin, which is typicallyfound on drier skin sites such as elbows and knees. Dry areas of theskin tend to have high microbial diversity and fewer populations thansebaceous sites. Use of beneficial bacteria, especially those modifiedvia the CRISPR-Cas and Cpf1 systems to enhance certain characteristicsof friendly beneficial bacteria, such as the amount of lipids produced,so as to remedy dry skin conditions, is one aspect of the presentinvention. Thus, in certain embodiments, taking resident species ofbacteria from a person's skin in a dry area and modifying such bacteriaso as to increase the amount of lipids produced by such bacteria, andpreferably enhancing particular other characteristics of such bacteriaso as to competitively inhibit other undesired bacteria from occupyingthose skin areas, is one method for enhancing the health of the person'sdry skin areas.

In the context of various embodiments, the use of antibiotic soapsshould be avoided so as to permit the beneficial bacteria as describedherein to generate positive conditions for beneficial bacteria growthand maintenance. Thus, to reduce the opportunity for more organisms todevelop a resistance to some of the substances, such as Triclosan, andthe removal of skin lipids alcohols and detergents by detergents andsoaps, such substances should be avoided when employing the beneficialbacteria formulations described herein, unless such use is performedprior to application thereof in a manner to reduce the number ofundesired bacteria first, followed preferably by a removal of suchantibacterial substances so that the desired bacteria can beadministered in a fashion that they prosper on the skin.

Damaged skin has also been found to be more frequently colonized byStaphylococcus hominis, Staphylococcus aureus, Enterococci and Candida.Thus, by topical application of effective amounts of the beneficialbacteria as described herein, especially those modified via the CRISPRsystems, it is possible to combat these undesired bacteria and promotethe health of an individual's skin microbiome.

Yet other aspects of the present invention relate to the skin on aperson's head, and in particular, relates to the maintenance of hairgrowth on the human scalp. Sulfation of minoxidil to generate ninoxidilsulfate is required to achieve the hair growth effect but minoxidilsulfate is unstable, and thus cannot be administered as is.Sulfotransferase enzyme can be used to sulfonate minoxidil to itstherapeutic form. In one embodiment of the invention, sulfotransferaseenzyme genes are provided via a CRISPR-Cas system such that modifiedbacteria are able to produce such enzyme in a manner that convertsminoxidil to its therapeutic form.

Psoriasis is a chronic, genetically based, immune-mediated inflammatorydisorder of the skin, present in about 2% of the world's population. Thecauses of psoriasis are poorly understood. The disease appears to resultfrom a combination of genetic and environmental factors. Certain skinbacteria, namely Staphylococcus aureus and Streptococcus pyogenes, playa role in the induction and maintenance of psoriasis. Modification ofthe microbiome of an individual, including not only the gut, but theskin microbiome, and especially employing a lotion that includes aprobiotic modified (via CRISPR-Cas systems) bacteria, as describedherein, is one effective way in which to treat Psoriasis and reduce itsnegative effects.

One objective of the present invention is to provide a method and systemto achieve a balanced microbial community in order to reduce oralleviate certain disorders. Personal care products related to microbialcommunities on the skin is one focus of various embodiments. Suchproducts include, in various embodiments, particular microorganisms thatcolonize the skin that include Staphylococcus epidermidis and othercoagulase negative staphylococci, as well as species of Corynebacteriumand Propionibacterium, all of which are preferably modified using theCRISPR-Cas or Cpf1 systems to delete certain virulence factors and toinclude beneficial traits, such as lipid production to facilitatemoisturizing characteristics of particular formulations.

Sebaceous sites, such as the forehead, have the lowest diversity, andPropionibacterium species are the most dominant organisms at such sites.On the other hand, moist areas (e.g., armpits, navel, groin) constitutehigher diversity of microbiota, with Staphylococcus and Corynebacteriumspecies as the predominant members. Moreover, skin sites with greaterbacterial diversity (e.g., forearm, hand, buttock) can harbor diversityas high as or higher than that of the gut microbiome. Acidic conditionsresulting from sebum degradation discourages pathogens from invading andestablishing in the skin. Personal hygiene is another environmentalfactor that has a direct effect on the skin's microbial flora. Soaps,makeup, and skincare products (e.g., moisturizers) alter skin conditionsthat in turn may influence the types of microbes residing on the skin.Among the host factors are age, sex, and anatomic sites. Skin microbiotadiffer among various age groups, with significantly different bacterialcommunities between the youngest and the oldest groups. A newbornacquires resident bacteria on the skin soon after birth, and theircomposition is affected by birth delivery methods. Hormonal changesduring puberty stimulate the growth of lipophilic (or lipid-loving)bacteria due to sebum production. Physiological changes and anatomicdifferences also contribute to microbial community variance betweengenders.

Certain skin diseases develop when altered lipid composition andorganization occurs. An example is acne, an inflammatory malady thataffects 80% of adolescents in the U.S. The change of lipid compositionduring puberty encourages lipophilic organisms, such asPropionibacterium acnes, to proliferate. As these bacteria derive energyfrom metabolizing fatty acids in the sebum, a variety of enzymes aresecreted that injure the tissue lining of sebaceous glands.

S. aureus is one of the most commonly cited skin pathogens, and it isresponsible for several cutaneous infections such as impetigo,furuncles, subcutaneous abscesses, ulcers, and other more serioussystemic infections when penetrating into the blood stream (e.g., toxicshock syndrome). Various embodiments of the present invention involvethe modification of S. aureus to reduce its virulence factors and tootherwise enhance its beneficial characteristics, especially in terms ofthe amount of moisturizing compounds produced by such bacteria afterbeing modified via a CRISPR-Cas system to include genes for desiredemollients, such as lipids.

Atopic dermatitis is a chronic and intensely inflammatory skin disorderthat has more than doubled in industrialized countries in the past threedecades without a clear cause. Atopic dermatitis patients frequentlyacquire cutaneous infections with S. aureus as the main colonizingorganism. Disease severity and bacterial diversity are related, withatopic dermatitis being most severe when community diversity is low, andas the modified microbiota as described herein increase in number, alevel of diversity similar to those of healthy skin may be attained.

Staphylococcus species have been implicated in both impaired woundhealing and leg ulcers. Thus, in certain other embodiments, modificationof such bacteria via CRISPR-Cas systems to reduce its virulence factors,and the subsequent use of competitive inhibition to reduce thepopulation of non-modified bacteria of the same species, provides a wayto address the issues long experienced by such bacteria impairing woundhealing and causing leg ulcers.

Neonatal skin reveals an abrupt wave of highly activated Treg cellsaccumulating in the tissue during the first weeks of life. Selectiveinhibition of Treg cell migration into skin, and during this periodcompletely abrogates commensal-specific tolerance. One aspect of thepresent invention is directed to appreciating that there is a specificwindow of time that is required for establishing a healthyhost-commensal relationship on the skin. Certain embodiments of thepresent invention are directed to providing beneficial bacteria, namelyparticular bacteria modified via CRISPR-Cas systems so as to generatedesirable immune functionality of a newborn by exposing such newborn toparticular species of bacteria and other microbes that trigger normaldevelopment of the newborn's immune system.

S. epidermidis is a prevalent commensal bacterial species on human skin.Skin bacterial antigens are recognized by the adaptive immune systemacross an intact skin barrier. Skin commensal bacteria influencecutaneous immunity without causing tissue inflammation. Because ahost-commensal relationship is formed immediately after birth, oneaspect of the present invention relates to the appreciation that themechanisms required for establishing tolerance are preferentially activeduring this period of time, such that administration of the variousdifferent and beneficial microbes to an infant's skin in the first 24hours of life may be critical in the infant from developing an entirehost of immune related diseases in later life.

Treg cells generated in neonatal life have the potential to promoteself-tolerance and tolerance to commensal antigens. The phenomenon of awave of Treg cells into neonatal tissue is unique to skin. Skin-barrierfunction influences more than local immunity and skin colonizationresults in commensal-specific T cells that are found both locally andsystemically, and thus, maintaining a healthy microbe-host immune dialogin skin is important for both systemic and tissue-specific immunehomeostasis. Altering the composition of skin commensal microbiota inthe neonatal period can limit the opportunity to establish tolerance toa wide array of microbial antigens, resulting in chronic tissueinflammation. There are a variety of chronic inflammatory diseases ofthe skin that form abnormal anticommensal immune responses. Thecomposition of the cutaneous microbiome in neonatal life has formativeeffects on the adaptive immune response to commensals, and understandingand manipulating the microbe is one aspect of the present invention withthe objective being to establish modifications to an individual's skinmicrobiome to achieve enduring health implications.

The skin microbiota is dependent on the body site and the colonizationof bacteria is dependent on the physiology of the skin site. Specificbacteria are associated with moist, dry and sebaceous microenvironments,with bacterial diversity lowest in such sebaceous sites.Propionibacterium spp. a lipophilic bacteria, is the dominant organismsin such sebaceous areas.

Certain bacteriophages are suitable for incorporating into certainantiperspirant and deodorant compositions, and are effective andspecific in fighting against bacteria causing odor without irritatingthe skin or damaging the germs in the flora of the skin which have apositive effect. According to the invention, cosmetic and/orpharmaceutical compositions suitable to deodorizing the body contain, ina cosmetic or dermatologically acceptable carrier, at least onedeodorant or antiperspirant active substance and at least onerepresentative from the group of bacteriophages which are effectiveagainst at least one of the following: Staphylococcus aureus and/orStaphylococcus hominis and/or Corynebacterium tuberculostearicum and/orAnaerococcus octavius and/or Staphylococcus lugdunensis and/orFinegoldia magna and/or Corynebacterium amycolatum and/orCorynebacterium afermentans and/or Staphylococcus epidermidis and/orStaphylococcus capitis and/or Staphylococcus haemolyticus and/orPropionibacterium avidum and/or Corynebacterium kroppenstedtii and/orpeptinophilus spec. Each of such bacteria can be modified usingCRISPR-Cas systems to reduce the production of malodorous components andto provide microbes that out compete wild type bacteria of the samespecies.

One particular aspect of the present invention is directed to decreasingbody odor by inhibiting Staphylococcus hominis that is involved in bodyodor formation. Thus, certain embodiments are directed to a deodorantcomposition or antiperspirant composition for the reduction of the bodyodor, involving applying to a person's skin a bacterial formulation thatincludes bacteria that generate lactic acid in combination withphysiologically acceptable salts, with sodium salt most preferred. Evenmore preferred embodiments involve the modification of Staphylococcushominis to reduce if not eliminate virulence factors of the species, andemploying such modified bacteria to competitively inhibit non-modifiedstrains, thus providing a way for an individual to reduce body odor(e.g. by at least 20%, more preferably at least about 50% and mostpreferably 80%) by inoculation of certain portions of their body, namelytheir armpits, with the modified bacteria. The sweaty odor of humans iscomprised of acids, thiols and steroids. Sulfur compounds in a person'smouth are similar to the thiols present in armpit malodors. Thus,reducing the expression of thiols in particular bacteria by CRISPR-Casand/or Cpf1 systems is implicated in various embodiments of the presentinvention.

Propionibacterium, and P. acnes in particular, are dominant organisms innormal skin. One aspect of the present invention is directed to theperhaps the anti-intuitive fact that maintenance of a robust populationand colony of such bacteria on an individual's skin plays a protectiverole that preserves and maintains normal skin health. Various illness ofindividuals occurs when P. acnes is displaced by more aggressiveorganisms. Thus, one aspect of the present invention is directed toCRISPR-Cas modified bacteria that have particular advantages incompetitive competition with wild type strains, such as being providedwith certain genes that render their colonization of the skin slightlymore preferred than wild type strains.

In some embodiments, genetic modifications are made in a Cutibacteriumacnes porphyrins gene, a CAMP-factor (CAMP1, CAMP2, CAMP3, CAMP4),Hyaluronate lyase (HYL-IB/II, HYL-IA), Lipases (GehA, GehB),Haemolysins, Sialidases, Endoglycoceramidases,Endo-β-N-acetylglucosaminidase, Dermatan sulphate adhesin (DsA1, DsA2),Proline-Threonine Repeats (PTRs) or in any virulence factors included onthe acne associated genomic loci 1, 2, 3 (plasmid), 4 such as a tightadhesion locus (tad), Streptolysin S-associated genes (sag),nonribosomal peptide synthetases (NRPS) as described in Tomida et al.sup.5, which is hereby incorporated by reference.

In one embodiment, the bacterial origin of replication is functional inPropionibacterium and Cutibacterium more specifically inPropionibacterium freudenreichii and Cutibacterium acnes and is selectedfrom the group consisting of pLME108, pLME106, p545, pRGO1, pZGX01,pPG01, pYS1, FRJS12-3, FRJS25-1, pIMPLE-HL096PA1, A_15_1_R1.

In a particular embodiment, the phage origin of replication is fromPropionibacterium phages: BW-like phages such as Doucette, B22, E6, G4,BV-like phages such as Anatole, E1, B3, BX-like phages such as PFR1 andPFR2, filamentous B5 phage, BU-like phages (Cutibacterium acnes phages).

In one embodiment, the targeted bacteria are Cutibacterium acnes morespecifically the acne related Cutibacterium acnes from the phylogroupIA1 or RT4, RT5, RT8, RT9, RT10 or Clonal Complex (CC) CC1, CC3, CC4,more specifically the ST1, ST3, ST4.

Certain embodiments of the present invention encompass the treatment ofdiseases and metabolic disorders caused by bacteria. The diseases ordisorders caused by bacteria may be selected from the group consistingof skin chronic inflammation such as acne (acne vulgaris), progressivemacular hypomelanosis, abdominal cramps, acute epiglottitis, arthritis,bacteraemia, bloody diarrhea, botulism, Brucellosis, brain abscess,cardiomyopathy, chancroid venereal disease, Chlamydia, Crohn's disease,conjunctivitis, cholecystitis, colorectal cancer, polyposis, dysbiosis,Lyme disease, diarrhea, diphtheria, duodenal ulcers, endocarditis,erysipelothricosis, enteric fever, fever, glomerulonephritis,gastroenteritis, gastric ulcers, Guillain-Barre syndrome tetanus,gonorrhoea, gingivitis, inflammatory bowel diseases, irritable bowelsyndrome, leptospirosis, leprosy, listeriosis, tuberculosis, LadyWidermere syndrome, Legionaire's disease, meningitis, mucopurulentconjunctivitis, multi-drug resistant bacterial infections, multi-drugresistant bacterial carriage, myocarditis, myonecrosis-gas gangrene,Mycobacterium avium complex, neonatal necrotizing enterocolitis,nocardiosis, nosocomial infection, otitis, periodontitis, phalyngitis,pneumonia, peritonitis, purpuric fever, Rocky Mountain spotted fever,shigellosis, syphilis, sinusitis, sigmoiditis, septicaemia, subcutaneousabscesses, tularaemia, tracheobronchitis, tonsillitis, typhoid fever,ulcerative colitis, urinary infection, whooping cough, NonalcoholicFatty Liver Disease (NAFLD), Nonalcoholic steatohepatitis (NASH).

Staphylococcus epidermidis is with Cutibacterium acnes one of the twomost prevalent and abundant commensal bacteria on the human skin. Assuch it has been shown to prevent colonization by pathogenic bacterialike its close relative Staphylococcus aureus, prevent skin cancer oralso modulate the human immune system. However, it is also a growingconcern due to its opportunistic pathogenic characteristic and itsgrowing resistance to antibiotics. These pathogenic traits of S.epidermis might be encoded on specific virulence genes or cluster andsome of these might spread across strains by horizontal gene transfer.

The loss of Propionibacterium presence on a person's skin is indicativeof psoriatic disease and thus, one objective in various embodiments isto limit or reduce numbers of over-represented organisms typically foundin disease states on the skin and to increase populations of commonlyoccurring resident microbes that are diminished in disease. Such aprobiotic approach is effective in addressing long unsolved skinconditions.

Commensal microorganisms that comprise the human microbiota are notsimply passengers in the host, but actually drive certain hostfunctions. One aspect of the present invention is to introducebeneficial bacteria to the skin, where such bacteria may not typicallyhave a real opportunity to survive and prosper. Thus, in severalembodiments, two or more bacteria are employed with each providingaspects of an environment such that another bacteria can survive. Forexample, one bacteria may provide an acid producing ability that anotherbacteria needs to prosper. Both bacteria can then depend on each otherfor survival—and if either is absent, then the other is eliminated. So,for example, in certain embodiments, formulations and methods ofadministration of skin microbes are designed to that certain bacteriapossess an antibiotic sensitivity that is only not expressed when theanother bacteria is present—such that if both are not there, then theyeach will die. This type of synergism is preplanned and calculated toprovide at least two different types of bacteria, in some embodiments atleast three types of bacteria, and in other embodiments at least fourbacteria species modified so that they are co-dependent upon each otherfor a critical factor in each other's survival, such that they all needto be present on the skin at the same time to co-survive. In such amanner, coordination of selection of bacterial species can be used tofoster the desired diversity of bacteria that has been appreciated asbeing beneficial to the overall health of a skin microbiome.

In certain embodiments, CRISPR-Cas systems are employed to modify theadhesion characteristics of particular bacteria, namely adhesions, whichare molecular parts of their capsules, fimbriae, and cell walls thatattach to a host surface. In certain embodiments, modifications may bemade to enhance the adherence of certain desired bacteria to particulartissues so as to competitively inhibit the attachment of other undesiredbacteria to such tissues.

Pathogenic bacteria display various levels of host specificity ortropism. While many bacteria can infect a wide range of hosts, certainbacteria have strict host selectivity for humans as obligate humanpathogens. Various aspects of the present invention are directed to theemployment of host specific bacteria that are modified via theCRISPR-Cas and/or Cpf1 systems in a manner that does not pose a threatto various other species, thus reducing the threats that might otherwisebe presented if large-scale modifications of bacteria are modified byCRISPR systems.

Certain bacteria are highly adapted to the human environment and displaystrict host selectivity for humans, including Haemophilus influenzae,Helicobacter pylori, Neisseria gonorrhoeae, Neisseria meningitidis,Mycobacterium leprae, Salmonella typhi, Streptococcus pneumoniae,Streptococcus pyogenes, Vibrio cholerae and Treponema pallidum. Oneaspect of the present invention relates to the modification ofhuman-specific pathogens to reduce virulence factors of such bacteriausing one of a CRISPR-Cas or Cpf1 system. Other embodiments include theenhancement of selective bacteria with respect to their ability to growand colonize the human skin by competitively inhibit other undesiredspecies. By rendering such human-specific bacteria, many considered tobe pathogenic, less harmful to humans, one aspect is directed toemploying such modified bacteria to outcompete and thus competitivelyinhibit the colonization of non-modified bacteria on a person's skin.

N. gonorrhoeae and N. meningitidis are two human pathogens within thegenus Neisseria. N. gonorrhoeae is the causative agent of gonorrhea(pelvic inflammation), a sexually transmitted disease. N. meningitidiscauses invasive infections, such as septicemia and meningitis. Bothpathogens have strict host tropism for humans. Like many otherhuman-specific pathogens, the narrow host specificity of thesepathogenic bacteria renders them suitable for modification to make themless pathogenic, and at least to reduce the population of wild type morevirulent strains. In one embodiment, CRISPR-Cas systems are used toexcise particular virulence factors of particular bacteria, such asNeisseria, preferably affecting one or more surface-exposed proteinsassociated with the human specificity of pathogenic Neisseria.

The present inventor contends that S. aureus colonization is a cause ofvarious diseases, and thus its reduction or near elimination results inan amelioration of the clinical symptoms. Staphylococcus aureus isresponsible for a variety of diseases ranging from minor skin infectionsto life-threatening systemic infections, including endocarditis andsepsis, and is a major cause of community- and hospital-acquiredsepticemia. Atopic dermatitis (AD) is a disease of skin microbiotadysbiosis with S. aureus, interventions that reduce S. aureuscolonization on the skin of AD patients is one objective of the presentinvention. There is an existing problem with respect to antibioticresistance of S. aureus microbiologists are now facing a challenge todesign strategies decreasing S. aureus skin load. S. aureus causes themajority of bacterial skin infections, including some historicallycaused by streptococcal species. Bacterial skin infections can beclassified as primary or secondary and as an initial episode or arecurrence. Primary infections manifest in normal, intact skin. Examplesinclude impetigo, cellulitis, folliculitis, or furunculosis. Secondaryinfections manifest in conditions with an impaired skin barrier. Commonexamples include atopic dermatitis, bites, burns, and wounds. In atopicdermatitis, S. aureus colonization is common and secondary S. aureusinfections are a major concern. Patients with atopic dermatitis are atrisk for secondary infections due to impaired physical barrier function,colonization with pathogenic bacteria, and alterations to the skinmicrobiome. An additional risk for infection relates to deficiencies inthe antimicrobial defenses of the skin.

In one embodiment, S. aureus is modified via CRISPR systems to rendersuch bacteria antibiotic sensitivity and such sensitive culture is thenpurposefully employed to populate the skin of an individual, thuscompetitively inhibiting the residence of other S. aureus on theperson's skin. Removal of bacteria, including S. aureus, prior to suchre-population step is preferably employed so as to facilitate there-establishment of the modified S. aureus as a bacterial species,despite the appreciation that such species is not a typically desiredbacteria on a person's skin.

Worsening atopic dermatitis and smaller bacterial diversity are stronglyassociated. Thus, one aspect of certain embodiments relates toincreasing the diversity of bacteria on a person's skin via theapplication of formulations that include beneficial bacteria in amountsand with particular diversity of species so as to promote the health ofa person's skin in a manner to reduce the likelihood of atopicdermatitis. The application of such formulations is preferably conductedwithout the use of emollients containing antioxidant and antibacterialcomponents that may reduce microbiome diversity in atopic skin. As aboutone third of deaths in adults in the elderly are due to infectiousdisease, it is believed that the present invention provides an avenue toreduce the number of such deaths and to otherwise address thesignificant health issues related to skin ailments, including but notlimited to atopic dermatitis. One theory is that as a person ages, theirimmune system changes and is less robust in addressing bacterialinfections. By enhancing the microbiome of a person's skin as they age,it is believed that infections that would otherwise be encountered willbe avoided, or at least the frequency and severity of the same will bedecreased.

Staphylococcus aureus is a Gram-positive, commensal bacterium known toasymptomatically colonize the human skin, nares, and gastrointestinaltract. Colonized individuals are at increased risk for developing S.aureus infections, which range from mild skin and soft tissue infectionsto more severe diseases, such as endocarditis, bacteremia, sepsis,bacteremia, pneumonia and osteomyelitis. Staphylococcus aureus is one ofthe most important bacterial pathogens in hospital- andcommunity-acquired infections. Different virulence factors are requiredfor S. aureus to infect different body sites. Various aspects of thepresent invention are directed to modifying bacteria using theCRISPR-Cas and Cpf1 systems to reduce various virulence factors,including those involved in S. aureus infections, and by doing so,protecting individuals from one or more of the diseases related to suchbacterium.

Probiotics are believed to play a part in protecting skin againstphotoaging. Supplementation of a person's skin with particular bacteria,and preferably a diverse set of bacteria, even more preferably bacteriathat have been modified by using a CRISPR-Cas or Cpf1 system to reducevarious virulence factors of such bacteria, and/or to incorporate UVprotectant chemicals and proteins generated by the modified bacteria, isemployed to significantly enhance skin hydration, reduce epidermalthickening and transepidermal water loss, and to further protect theskin form harmful UV radiation. Supplementation of a person's skin withan effective amount of a Bifidobacterium strain, preferably modified viaCRISPR-Cas system to enhance UV protection, is one aspect of variousembodiments.

Yet another aspect of the invention is directed to a method via which abacterial containing lotion, gel or cream is administered topically toprovide a person with a diverse number and type of bacteria, especiallythose modified via CRISPR systems as described herein, and in such amanner, reduce the likelihood of skin infections.

Certain aspects of the present invention relate to a compositionincluding ammonia oxidizing bacteria to increase production of nitricoxide and/or nitric oxide precursors in close proximity to a person'sskin. More specifically, applying a composition of an ammonia oxidizingbacteria to skin during or after bathing to metabolize urea and othercomponents of perspiration into nitrite and ultimately into Nitric Oxide(NO) results in a natural source of NO. One aspect of the presentinvention causes topical nitric oxide release at or near the surface ofthe skin where it can diffuse into the skin and have local as well assystemic effects. This naturally produced nitric oxide can thenparticipate in the normal metabolic pathways by which nitric oxide isutilized by the body. Adding urea or ammonium salts to the skin providesadditional substrates that these bacteria utilize to form nitrite. Asused herein, the phrase near the surface is defined as adjacent to or inclose proximity to, but need not be in contact with the surface.

Prior to the advent of frequent bathing in hot water and soapsubstances, the skin on a human would develop a natural community ofmicroorganisms adapted to the skin environment. An abundant component ofhuman perspiration is urea. In soil, natural bacteria act upon urea andhydrolyze it to ammonia, which is then oxidized to nitrite, followed byrapid oxidation, by still other bacteria, to nitrate. In soil, allnitrogen containing compounds are ultimately degraded to nitrate. It isnitrate that most plants absorb as their nitrogen source. Underconditions of infrequent bathing, skin bacteria that can metabolize ureainto nitrite—thrive and proliferate. The resulting nitrite on the skinwhen dampened by additional perspiration at the normal sweat pH of 4.5would release Nitric Oxide (NO). Nitric Oxide is a small molecule thatdiffuses rapidly through the skin into the capillaries of the skin.Vasodilatation of these capillaries occur, as well as diffusion of NOinto the blood where it can be transported to other regions of the body.Dilatation of the capillaries at the skin surface enhances blood flowto, and hence heat loss from the skin during periods of exercise.

In certain embodiments, an ammonia oxidizing bacteria may be used andpreferably may have the following characteristics: ability to rapidlymetabolize ammonia and urea to nitrite and other NO precursors; nonpathogenic; non allergenic; non-producer of odoriferous compounds;non-producer of malodorous compounds; ability to survive and grow inhuman sweat; ability to survive and grow under conditions of high saltconcentration; and ability to survive and grow under conditions of lowwater activity. Natural bacteria can be used as well as bacteria whosecharacteristics have been altered through genetic engineeringtechniques, preferably via CRISPR systems as set forth herein. Whilesome skin bacteria species double every 20 minutes, ammonia-oxidizingbacteria, preferred in various embodiments of the present invention, aremuch slower growing, doubling only every 10 hours. Nitrosomonaseutropha, an ammonia-oxidizing bacteria is one preferred species toemploy on a person's skin to enhance the health of the skin and to avoidchemical use typically deemed required to thwart body odor.

Compositions of the present invention may take the form of a gel, acream, a lotion, an ointment, a solution, a solid “stick,” etc., thatcan be rubbed or sprayed onto the skin. Certain embodiments includewater, live cultured ammonia-oxidizing bacteria, disodium phosphate, andmagnesium chloride. Preferably, such compositions include selectedbacteria that have been modified via CRISPR-Cas or Cpf1 systems tospecifically target disease states and are used to reduce the effectsthereof. For example, particular compositions include liveLactobacillus, employing various strains that can also be found inedible compositions, such as probiotic yogurts and nutritionalsupplements. In preferred embodiments, there is an absence of sodiumlauryl sulfate, a potent detergent, which is preferably avoided as itcan remove your healthy bacteria. Other embodiments employBifidobacterium longum and Lactobacillus plantarum, both of which aremodified via the CRISPR systems as described herein. It is believed thatsuch Lactobacillus species reduce symptoms of eczema, especially whenmodified to encourage its growth and maintenance on the skin, wherenormally it is not found in abundance.

In still other embodiments, CRISPR systems are used to modify the generaPropionibacterium, Corynebacterium and Staphylococcus, and in particularS. epidermidis, which are among the most common groups on a person'sskin, with such modifications making such species more amenable togrowth on the skin, thus providing for competitive inhibition ofnon-modified bacteria on the skin. As one of skill in the art willappreciate, a suitable topical composition comprising a population ofthe above bacteria can be, in various embodiments, a cream, lotion,emulsion, gel, ointment, liquid or spray. In one embodiment, the topicalcomposition is formulated to provide at least about 10.sup.2 bacteriaper cm.sup.2. In another aspect, a method of treatment is provided,wherein a composition as described herein is topically applied to theskin and in certain embodiments, topically applying includes topicallyapplying to a mucosal surface (nasal, vaginal, rectal, oral surfaces) ofa person. A suitable lotion may also include amounts of sugars that thevarious Lactobacillus microorganisms may assimilate to survive andthrive. These sugars and life bacteria-supporting compounds are known tothose in the art and as otherwise referenced in various incorporatedwritings. In still other embodiments, pulverized compositions ofhelminth collections and bacteria preferably obtained from Amish-soils,may be employed in various administrative modes, including but notlimited to lotions, creams, and other topical applications.

Under the FD&C Act, a product's category is based primarily on intendeduse. For example, the term “cosmetics” is defined in section 201(i) ofthe FD&C Act in part as “articles intended to be rubbed, poured,sprinkled, or sprayed on, introduced into, or otherwise applied to thehuman body or any part thereof for cleansing, beautifying, promotingattractiveness or altering appearance”. Thus, various embodiments of thepresent invention are directed to cosmetic compositions that includemicroorganisms or fractions thereof that enhance the health of aperson's skin. Still other embodiments are directed to a shampoo and aconditioner having a sufficient amount of bacteria therein tobeneficially affect the scalp of the user. Yet other embodiments aredirected towards personal care products, including those that may helpto prevent disorders or restore health of the skin, such as, but notlimited to, particular cuticle formulations that are beneficial to thehealth of a person's nail beds on their hands and feet.

One aspect of the present invention is to determine a person'sparticular microbiome composition, compare it generally to thecharacteristics of the general population in the general area where suchindividual resides, and then address any differences in such microbepopulations differences on the particular person by providing particularmicrobe components, whether that be actual microbes themselves or theprovision of nutrients for particular microbes to outcompete othermicrobes, such that a population of microbes more like those of thegeneral population is achieved.

One basis from which several embodiments of the present invention relateinclude an appreciation that the general population of microbes in ahuman population in a particular area, for example in a city, will havecertain general characteristics. Individuals that possess microbepopulations on their skin that vary significantly from such generalpopulation of microbes are often suffering from maladies that aredirectly related to the imbalance of microbe populations on their skin.As such, one aspect of the present invention is to address suchimbalance by providing methods and compositions that bring theparticular microbe population of a particular individual back into therelative balance of microbe population as exists in the particular citywithin which such individual lives.

There are noted differences of microbe populations on an individual'sskin dependent on race, gender, age and living environment, particularlythe location of the person, e.g. whether they are in the tropics,northern climes, etc. These factors are taken into account whenaddressing the alteration of a particular person's microbiome so thatmodifications made to the individual's microbiome are adjusted toachieve roughly the same microbe population as would be predicted frommore general criteria of a hypothetical person having similar race,gender and age characteristics in a particular locale. In a way, suchconformance with respect to the microbiome in a community is abeneficial trait for such community, as well as for the individualsresiding therein. There has not been, prior to the present invention, amethod or system that addresses the imbalances that exist periodicallyin a community's overall microbiome so that issues arising fromindividuals who present personal microbiomes that present significantlydifferent microbe populations on their skin can be addressed by, forexample, reducing the type and number of bacteria on such person's skin,followed by purposeful exposure of such person's skin to a predeterminedpopulation of bacteria as described herein. By such exposing of suchperson to microbes and microbe enhanced compositions, one is able toachieve a more uniform overall microbiome of an entire community andenjoy the health benefits derived therefrom.

Various factors affect the microbial flora of the skin and they can begenerally categorized into host and environmental factors. Sebaceoussites such as the forehead have the lowest diversity, andPropionibacterium species are the dominant organisms. On the other hand,moist areas (e.g., armpits, navel, groin) constitute higher diversity ofmicrobiota, with Staphylococcus and Corynebacterium species as thepredominant members. Skin sites with greater bacterial diversity (e.g.,forearm, hand, buttock) can harbor diversity as high as or higher thanthat of the gut microbiome. The acidic condition resulting from sebumdegradation discourages pathogens from invading and establishing in theskin. Personal hygiene is another environmental factor that has a directeffect on the skin's microbial flora. Soaps, makeup, and skincareproducts (e.g., moisturizers) alter skin conditions that in turn mayinfluence the types of microbes residing on the skin. Host factors, suchas age, sex, and anatomic sites demonstrate that skin microbiota differamong various age groups. For example, acne, an inflammatory malady thataffects 80% of adolescents, relates to a change of lipid compositionduring puberty that encourages the proliferation of lipophilicorganisms, such as Propionibacterium acnes. As these bacteria deriveenergy from metabolizing fatty acids in the sebum, a variety of enzymesare secreted that injure the tissue lining of sebaceous glands. Inconjunction with activated immune responses, this results in a skincondition termed acne vulgaris. In contrast, younger children have ahigher abundance of Staphylococcus (S.) aureus, which are later replacedby lipophilic and other bacteria. Conversely, certain skin disorders,such as atopic dermatitis (or eczema), are more prevalent among childrenbut often resolve by adolescence and adulthood. S. aureus is one of manyskin pathogens responsible for several cutaneous infections such asimpetigo, furuncles, subcutaneous abscesses, ulcers, and toxic shocksyndrome. Burn victims whose epidermis have been destroyed are exposedto various assaults, with Gram positive bacteria (e.g., S. aureus) beingmain colonizers after a burn. A shift then occurs and Gram negativeopportunistic organisms predominate, some with virulent properties thatcan cause life threatening infections. Thus, in certain embodiments ofthe present invention, a particular formulation is provided thatincludes a diverse range of bacteria that have been preferably modifiedvia the CRISPR systems as described herein so as to establish aparticular population of diverse bacteria that enhance the healingenvironment of a burn victim's skin.

Atopic dermatitis, an inflammatory skin disorder that has more thandoubled in industrialized countries in the past three decades, istreated by administration of various formulations of the presentinvention. AD patients frequently acquire cutaneous infections with S.aureus as the main colonizing organism, with disease severity directlyrelated to low diversity of bacteria. Thus, to treat the same, promotionof diversity of bacterial species is achieved by administeringCRISPR-cas modified bacteria, including Staphylococcus species that havehad their virulence factors reduced so as to competitively inhibit thepopulation of other non-modified bacteria of the same species, oftenassociated with Chronic non-healing wounds. Provision of a diversepopulation of other beneficial skin microbes with such bacteria isbelieved to remedy such long term diseases. As dysbiosis in skinbacterial habitat is an indicator of unhealthy skin conditions, oneaspect of the present invention is directed to restoring homeostasis totreat certain skin diseases, including the administration of CRISPR-Casmodified bacteria that reduce the effectiveness of particular virulencefactors for the targeted bacteria and the encouragement of thepopulation of such enhanced and modified bacteria so as to maintain ahealthy and diverse population of the skin.

The many layers and structures of the skin serve as elaborate hosts tomicrobes, including a diversity of commensal and pathogenic bacteriathat contribute to both human health and disease. In severalembodiments, a formulation includes a diverse population of bacterialspecies based on a collection of various racial skin types and forparticular ethnic populations, such as an enhanced proportion ofEnhydrobacter for Chinese ancestry. Other formulations include bacterialformulations with Actinobacteria, Proteobacteria, Firmicutes,Bacterioidetes, in 40/30/20/10 ratios. Firmicutes (mainly Streptococcusand Staphylococcus), Enhydrobacter, Gordonia, and Actinobacteria (mainlyCorynebacterium and Propionibacterium) are preferably employed, againmodified via CRISPR-Cas and Cpf1 systems to reduce the virulence factorsnormally encountered in such bacteria. In various embodiments,CRISPR-Cas systems are employed to modify the following speciesbelonging to the genera Corynebacterium, Staphylococcus, Streptococcus,and Anaerococcus.

During the birthing process and subsequent exposure to the post-natalenvironment, the skin is colonized by a wide array of microbes.Knowledge of the skin microbiota has historically been limited toculture-dependent assays, although it is estimated that less than 1% ofbacterial species can be cultivated. Recent findings reveal a low levelof interpersonal consensus and an extremely dynamic microbiota thatfluctuates greatly in a short span of time. So changing a person'sbacterial skin composition is not something that should be viewed assomehow detrimentally affecting their health as such populations areseen to vary greatly in any event under natural conditions. Thus, oneaspect of the present invention relates to the modification of aperson's microbiota on their skin in a manner believed to enhance theoverall health of the skin, thus preventing diseases that may otherwiseinfect such person's skin. The diversity of the skin formulations as setforth herein include compositions that include at least the following(and particularly such species modified via CRISPR systems to reducetheir respective virulence factors and to enhance their abilities to outcompete other bacteria on a person's skin): Proteobacteria, of theJanthinobacterium, Serratia, Halomonas, Stenotrophomonas, Delftia, andComamonas genera; Actinobacteria, including species of the generaCorynebacterium, Kocuria, Propionibacterium, Microbacterium, andMicrococcus; Firmicutes, such as Staphylococcus or Clostridium species;and Bacteroidetes, including Sphingobacterium or Chryseobacteriumspecies. In other embodiments, a common core skin microbiome is derivedfrom healthy human subjects, and then such bacteria are enhanced viaCRISPR-Cas systems to remove virulence factors, prior to administeringthe modified bacteria to a person's skin for the purpose of improvingthe skin microbiome thereof.

The general Pseudomonas and Janthinobacterium (both pseudomonads;gram-negative bacilli, aerobic, non-spore forming, motile by means ofone or more flagella) are not typically thought of as skin microbesbased on culture assays. Pseudomonads (and other Gram-negative bacilli)have historically been labeled as secondary invaders of wounds, mostcommonly referring to the colonization of burns by Pseudomonasaeruginosa. Pseudomonads are found in soil, water, and decomposingorganic materials where there is a moist environment. Modification ofsuch bacteria so that it is less virulent and more readily reduced innumber due to CRISPR-Cas enhanced antibiotic sensitivities, forms oneembodiment of the present invention, and one that can be employed totreat wound infections to competitively inhibit non-modified strainsfrom infecting a wound.

Because skin cells turn over every 4 weeks, differentiating from stemcells deep within the epidermis and hair follicles, they eventuallyslough off from the upper layer as cornified (enucleated, dead) cells.The skin microbiome is vastly different from the gut microbiome, whichconsists primarily of members of Firmicutes and Bacteroidetes divisions.The skin is also different from the gut in that there is a low level ofinterpersonal variation of skin microbiomes, which is not the case ingut studies. Regardless, there is a low level of deep evolutionarylineage diversity, with only six of the more than 70 described bacterialdivisions associated with the skin, and approximately the same numberfor the gut, which compares to a vast array of bacteria in soil.

Many physiological functions are performed by skin microbiota.Proteobacteria, which dominates the skin microbiota, may be modified viaCRISPR-Cas and Cpf1 to achieve desired populations that lack virulencecharacteristics of wild type strains.

Still other embodiments relate to the adjustment of the microbiome viaair conditioning units, HVAC, etc units that propagate beneficialpopulations into a living environment of individuals. Certain figuresfrom the parent specification provide details as to how such microbescan be disseminated into a living space for humans, which affects bothrespiratory and skin microbiomes of individuals so exposed.

FDA acceptable limits for total (not pathogenic) microorganisms incosmetics are very low and are anticipated to be revised to permit theemployment of various embodiments of the present invention after arecognition that such products are safe and beneficial and addressvarious disease states. In preferred embodiments, bacteria that producevarious products are preferred, such as lactic acid, which serves as amoisturizing factor, hyaluronic acid which improves skin hydration andelasticity, and sphingomyelinase which generates ceramide for skinbarrier function. Whether such products are considered a drug or acosmetic under the FD&C Act, is something the present inventors believeis besides the point: the skin microbiome health is dependent upon theadministration of one or more of the compositions as set forth hereinand these will increase the benefits sought by numerous individuals.

Yet other embodiments include a topical lotion that comprises a mixtureof Lactobacillus johnsonii, and Bifidobacterium lognum bacteria. Suchuse of probiotic bacteriotherapy is employed in treating skin diseasesincluding eczema, atopic dermatitis, acne, and allergic inflammation orin skin hypersensitivity, UV-induced skin damage, wound protection, andas a cosmetic product. A topically applied composition that comprises apopulation of pre-selected bacteria comprising various non-pathogenicbacteria and CRISPR-Cas modified pathogenic bacteria is employed toestablish a resident population on a person's skin in order to enhancethe overall health of the person's skin.

In one such embodiment, a formulation comprises a mixture of variousamounts of the following: Bifidobacterium lognum, B. infantis BCRC14602; Prevotella; Ruminococcus, Bifidobacterium infantis, Lactobacillusacidophilus, Bacteroides fragilis, B. longum by Infantis isolate UCD272;B. infantis BCRC; B. longum by Infantis, AY151398; and Lactobacillusruminis. Other embodiments include compositions directed to thetreatment of sensitive skin, using, as active agent, a combination of aLactobacillus paracasei or casei microorganism and a Bifidobacteriumlongum or Bifidobacterium lactis microorganism.

Certain microorganisms are known to have a beneficial action on the skinmembrane by maintaining a slightly acidic environment. Thus, in certainembodiments, such an acidic environment is first established on the skinand then one maintains beneficial endogenous flora on the skin such thatthe microflora participates in maintaining a desired physio-chemicalbalance of the skin while not favoring the development of pathogenicmicroorganisms on the skin surface. The benefits of the skin conditionare selected from the group consisting of improving skin appearance,improving skin feel, increasing the thickness of one or more layers ofthe skin, increasing the elasticity of the skin, increasing theresiliency of the skin, increasing the firmness of the skin, reducing anoily appearance of the skin, reducing a shiny appearance of the skin,reducing a dull appearance of the skin, increasing a hydration status ofthe skin, increasing a moisturization status of the skin, reducing anappearance of fine lines, reducing an appearance of wrinkles, improvingskin texture, improving skin smoothness, improving skin exfoliation,improving skin desquamation, plumping the skin, improving skin barrierproperties, improving skin tone, reducing an appearance of redness,reducing an appearance of skin blotches, improving the brightness of theskin, improving the radiancy of the skin, improving the translucency ofthe skin.

A subject of the invention is also the topical use of an effectiveamount of at least one probiotic microorganism according to theinvention, especially of the Lactobacillus and/or Bifidobacterium sp.Genus, and in particular of the Lactobacillus paracasei ST11 strain, toreduce the likelihood of seborrhoeic dermatosis associated with oilyskin or skin with an oily tendency. Microorganisms suitable for thisaspect of the invention include an ascomycetes, such as Saccharomyces,Yarrowia, Kluyveromyces, Torulaspora, Schizosaccharomyces pombe,Debaromyces, Candida, Pichia, Aspergillus and Penicillium, bacteria ofthe genus Bifidobacterium, Bacteroides, Fusobacterium, Melissococcus,Propionibacterium, Enterococcus, Lactococcus, Staphylococcus,Peptostrepococcus, Bacillus, Pediococcus, Micrococcus, Leuconostoc,Weissella, Aerococcus, Oenococcus and Lactobacillus, and mixturesthereof.

As ascomycetes is particularly suitable for particular embodiments ofthe present invention, one may desire the use of Yarrowia lipolytica andKluyveromyces lactis, as well as Saccharomyces cerevisiae, Torulaspora,Schizosaccharomyces pombe, Candida and Pichia, all of the samepreferably modified via CRISPR-Cas or Cpf1 systems to reduce virulencefactors associated with the same. Specific examples of probioticmicroorganisms also suitable for the invention include: Bifidobacteriumadolescentis, Bifidobacterium animalis, Bifidobacterium bifidum,Bifidobacterium breve, Bifidobacterium lactis, Bifidobacterium longum,Bifidobacterium infantis, Bifidobacterium pseudocatenulatum,Lactobacillus acidophilus (NCFB 1748); Lactobacillus amylovorus,Lactobacillus casei (Shirota), Lactobacillus rhamnosus (strain GG),Lactobacillus brevis, Lactobacillus crispatus, Lactobacillus delbrueckii(subsp bulgaricus, lactis), Lactobacillus fermentum, Lactobacillushelveticus, Lactobacillus gallinarum, Lactobacillus gasseri,Lactobacillus johnsonii (CNCM I-1225), Lactobacillus plantarum,Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillusalimentarius, Lactobacillus curvatus, Lactobacillus casei subsp. casei,Lactobacillus sake, Lactococcus lactis, Enterococcus (faecalis,faecium), Lactococcus lactis (subsp lactis or cremoris), Leuconostocmesenteroides subsp dextranicum, Pediococcus acidilactici,Sporolactobacillus inulinus, Streptococcus salvarius subsp.thermophilus, Streptococcus thermophilus, Staphylococccus carnosus,Staphylococcus xylosus, Saccharomyces(cerevisiae or else boulardiO,Bacillus (Cereus var toyo or subtilis), Bacillus coagulans, Bacilluslicheniformis, Escherichia coli strain nissle, Propionibacteriumfreudenreichii, and mixtures thereof. In other embodiments, probioticmicroorganisms for use in the invention are derived from the group oflactic acid bacteria, such as, in particular, Lactobacillus and/orBifidobacterium. In particular, various embodiments use lactic acidbacteria such as Lactobacillus johnsonii, Lactobacillus reuteri,Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus casei orBifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum,Bifidobacterium animalis, Bifidobacterium lactis, Bifidobacteriuminfantis, Bifidobacterium adolescentis, Bifidobacteriumpseudocatenulatum, and mixtures thereof. Most preferably for particularembodiments, CRISPR modified bacteria of the following are employed:Lactobacillus johnsonii, Lactobacillus paracasei, Bifidobacteriumadolescentis and Bifidobacterium longum, respectively depositedaccording to the Treaty of Budapest with the Institut Pasteur (28 rue duDocteur Roux, F-75024 Paris cedex 15) on 30 Jun. 1992, 12 Jan. 1999, 15Apr. 1999 and 15 Apr. 1999 under the following designations: CNCM1-1225, CNCM 1-2116, CNCM 1-2168 and CNCM 1-2170, and theBifidobacterium lactis (Bb 12) (ATCC27536) or Bifidobacterium longum(BB536) genus. The Bifidobacterium lactis (ATCC27536) strain can beobtained from Hansen (Chr. Hansen A/S, 10-12 Boege Alle, P.O. Box 407,DK-2970 Hoersholm, Denmark); Lactobacillus paracasei ST11 straindeposited according to the Treaty of Budapest with the Institut Pasteur(28 rue du Docteur Roux, F-75024 Paris cedex 15) on 12 Jan. 1999 underthe designation CNCM 1-2116, and/or a fraction thereof and/or ametabolite thereof.

According to one variant embodiment, the invention relates to the use,in addition to a first probiotic microorganism, as defined above,especially of the Lactobacillus and/or Bifidobacterium sp. genus, of atleast an effective amount of at least a second microorganism, distinctfrom said first microorganism. Such a second microorganism may be anascomycetes, such as Saccharomyces, Yarrowia, Kluyveromyces,Torulaspora, Schizosaccharomyces pombe, Debaromyces, Candida, Pichia,Aspergillus and Penicillium, bacteria of the Bacteroides, Fusobacterium,Melissococcus, Propionibacterium, Enterococcus, Lactococcus,Staphylococcus, Peptostrepococcus, Bacillus, Pediococcus, Micrococcus,Leuconostoc, Weissella, Aerococcus, Oenococcus, Lactobacillus orBifidobacterium genus, and mixtures thereof.

In other embodiments, CRISPR-Cas and or Cpf1 systems are used to modifyat least one of Enterobacter aerogenes, Acinetobacter baumannii, andKlebsiella pneumoniae, which are three gram negative bacteria commonlyfound on the skin, and which utilize fatty acids in a manner thataffects bacterial phenotype. The modifications to such bacteria includethose effective in enhancing the beneficial traits of such bacteria fora person's skin and the reduction of respective virulence factors of thebacteria. In such a manner, one aspect of the present invention is tomaintain a microbiome in a healthy, balanced state and/or returning amicrobiome to a balanced state by providing certain desirablemicroorganisms with sufficient nutrients to thrive, and therebyoutcompete and/or kill the undesirable bacteria. It has been found thatCorynebacterium jeikeium (“C. jeikeium”), Staphylococcus epidermidis(“S. epidermidis”), and Propionibacterium acnes (“P. acnes”), present onboth the face and forearms of humans, can be used to address dry skinconditions and diseases on such tissues. Modifications of virulencefactors of pathogenic bacteria associated with such conditions, as wellas combining such modified bacteria with other commensal microorganisms,is one aspect of the present invention. Such bacteria include:Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria,Propionibacteria, Corynebacteria, Actinobacteria, Clostridiales,Lactobacillales, Staphylococcus, Bacillus, Micrococcus, Streptococcus,Bacteroidales, Flavobacteriales, Enterococcus, and Pseudomonas.

One particular aspect of the present invention is directed to a methodand system for reducing the likelihood that one will acquire multiplesclerosis (MS). To avoid such disease, the avoidance of a skincontacting soluble toxin is achieved by establishing a population ofbeneficial bacteria, as described herein, on a person's skin, such thatClostridium perfringens, a gram positive, spore forming anaerobe, whichproduces the toxin required for the initiation of MS, is avoided.Certain aspects of the present invention are directed to a system andmethod to protect a person from getting MS and/or to treat a person thathas multiple sclerosis by interfering with epsilon toxin (ETX) ofClostridium perfringens type B or type D by interfering with the ETXinteracting receptor, and in particular, by employing CRISPR-Cas and/orCpf1 systems to modify bacteria in a manner to reduce the ability ofClostridium perfringens to produce effective toxins that can trigger MS.Use of CRISPR-Cas systems to provide inhibitors of such toxin, such asthe inclusion of genes that express antibodies thereto, is oneembodiment of the present invention. Competitive inhibition ofClostridium perfringens by using an effective amount of a probioticsupplement which contains a bacterial strain that outcompetes suchnon-modified species, is one way to provide a treatment regimen, and oneof skill in the art will appreciate the many desirable strains by whichto accomplish this objective, including but not limited to thefollowing: Lactobacillus acidophilus, L. bulgaricus, L. casie, L.fermentum, L. plantarum, Rhodopseudomonas palustris, Saccharomycescerevisiae, and Streptococcus thermophiles. To render such competitiveinhibition effective, it is suggested that in preferred embodiments, acourse of antibiotics is taken first to reduce the numbers of C.perfringens, preferably employing one or more of penicillin, ampicillin,amoxicillin, metronidazole, erythromycin, and tylosin, prior toinoculation (for some skin application, rubbing of a lotion or gel onone's skin) of the person's microbiome with one or more of the probioticbacteria listed herein, and in particular, those bacteria modified bythe CRISPR-Cas and/or Cpf1 systems described herein.

Various embodiments of the present invention are directed to a methodfor reducing the likelihood of the onset of a disease by administeringto a subject a therapeutically effective amount of a compositioncomprising a probiotic microorganism, rather than attempting to alterthe eucaryotic genome of the individual. It is believed that by merelymodifying a person's microbiome, whether it be their gut, oral or skinmicrobiome, it is possible to treat, if not protect such individualsfrom a vast array of previously devastating diseases of man. Forexample, Helicobacter species have been associated with enhancedcarcinogenesis including liver cancer, colon cancer, and mammarycarcinoma. Probiotic formulations containing lactic acid bacteria havebeen shown to reduce the incidence of chemically mediated hepatocellularcarcinoma and colon cancer. Bacteria that have been modified using aCRISPR-Cas system to purposefully excise or interfere with virulencefactors of particular pathogenic bacteria, and the employment of suchmodified bacteria to adjust the population of a person's microbiome, isan effective way to treat a vast number of historically difficultdiseases.

Certain aspects of the present invention are directed to modifying aperson's intestinal, oral or skin microbiota using specific combinationsof prebiotics, probiotics and/or anti-biotics to establish a definedmicrobiota that can treat and/or reduce the likelihood that individualswill experience various diseases. For example, various embodiments ofthe present invention are directed to averting or reducing thelikelihood of cancer by employing bacteria modified to address p53deficiency. In such a manner, rather than treating human cells and theconsequent issues surrounding genetic manipulation of human cells fortreatments of cancer, the present invention provides a method and systemthat employs the microbiome of a person, whether than be oral, gut orskin, or a combination thereof, to treat cancer by increasing the levelof p53 to take advantage of the role of such protein in the progressionof various cancers. Provision of modified bacteria as described hereinto pre-treat a person prior to a cancer treatment, such as radiation,can also be used to lessen the otherwise detrimental effects of theradiation treatment. Moreover, after such treatments, provision of suchmodified bacteria to restore the person's microbiomes, whether they beoral, skin or intestinal, is one aspect of the present invention. Use ofmodified skin bacteria to treat melanoma is one aspect of the presentinvention, thus providing a way to treat skin cancer by providingessential compounds to reduce the spread and health of cancer cellswhile at the same time, enhancing the growth and propagation ofbeneficial bacteria, especially those modified as described herein via aCRISPR system.

The balance between health and disease is imperiled by infections. Whenimmunity is lowered, the human body is less able to eradicate cancercells, which would otherwise be kept in check. In certain embodiments, amushroom component is also employed to achieve desired health effects.For example, in various embodiments, the mushroom mycelium is used toprotect against viruses that cause disease in humans, such as thosemushrooms derived or obtained from Antrodia, Fomes, Fomitopsis,Ganoderma, Inonotus, Schizophyllum, Phellinus, Piptoporus, Trametes andother taxa in the Polyporaceae. Ethyl alcohol/water extractiontechniques are employed on living mycelium to obtain antiviral compoundsand that are effective to reduce viruses that cause inflammation andimmune deactivation which are contributory to oncogenesis. Such extractsreduce the pathogenicity of viruses and by doing so, reduce cancer riskand also significantly enhance the benefits of other anticancer drugs toincrease the quality of life of cancer patients. Used in combinationwith the various other aspects of the present invention, including thebeneficial modified bacterial species as described herein, a person'soverall health is improved by reducing the chances of infection,inflammation and cancer, by improving and adjusting the microbiome ofindividuals and by having certain mushroom derived compoundsadministered, (some of which can be inserted into the genome of bacteriavia the CRISPR-Cas system) such that beneficial compounds areadministered to individuals to prevent and treat various diseases, suchas but not limited to, cancer.

In particular embodiments, a method of the present invention involves amethod of improving the health of a person's skin microbiome byidentifying a skin region to be treated in terms of age, ethnicity,region of the body and age of the person and then applying a skincommensal prebiotic agent adapted to address the skin region; whereinthe prebiotic comprises at least one microbe that has been modified by aCRISPR-Cas or Cpf1 system to add or delete a gene that enhances thehealth of a person's skin.

Other embodiments include a method of improving the health of a person'sskin microbiome, comprising: providing a first type of bacteria to aperson's skin that produces an agent that another second bacterialspecies requires for growth; after applying said first bacteria to theskin of a person, then applying the second bacteria to the person'sskin, wherein both the first and the second bacteria comprise at leastone microbe that has been modified by a CRISPR-Cas or Cpf1 system to addor delete a gene that enhances the health of a person's skin. In stillothers, the virulence factor of the first bacteria is modified viaCRISPR-Cas to impede the interaction of bacterial adhesions andkeratinocyte receptors. One can modify the expression of at least onegene by employing a CRISPR-Cas system to decrease the pathogenesis of askin infection. Moreover, one can employ a second bacteria whose growthon a person's skin is enhanced by at least 2-fold when in the presenceof the first bacteria, wherein the second bacteria is modified viaCRISPR-Cas to have an essential growth required component deleted fromits genome, and wherein the first bacteria has been modified viaCRISPR-Cas to add the same essential growth component that the secondbacteria requires for growth.

Existing antibiotic therapies non-specifically kill the majority ofskin-residing bacteria, disrupting the homeostasis of skin residentmicroflora. For example, benzoyl peroxide (BPO) is one of the mostfrequently used topical medications. BPO strongly suppresses the growthof S. epidermidis. S. epidermidis contributes to the skin residentmicroflora-based defense of the skin epithelium. The imbalance ofmicroflora is believed by the present inventor to contribute to thepathogenesis of skin inflammatory diseases, such as atopic dermatitis,rosacea and acne vulgaris etc. Thus, in various embodiments, suchantibiotic therapies are not employed but instead, beneficial bacteriaare administered to a person's skin in a manner that beneficial resultsare achieved (e.g. reduction in malodors, generation of desired odors bybacterial production of scents, etc.) CRISPR-Cas systems are preferablyemployed to modify species of bacteria already found on an individual'sskin such that the disturbance of the “normal” population of aparticular person is not disturbed in a fashion that could lead todisease or discomfort.

Various embodiments include providing two or more bacteria species thatare normally found on a person's skin, and modifying the same to removevirulence factors via CRISPR; including in such bacteria beneficialgenes for the production of emollients, lipids, scents, etc. and usingcompetitive inhibition to foster the growth of bacteria purposefullyexposed to the skin surface so that pathogenic bacteria are notpermitted to establish and grow. In certain embodiment, CRISPR isemployed to insert a gene for the production of tomatidine in a bacteriasuch that, especially, in the gut microbiome, but preferably also in theoral and skin microbiome, tomatidine is expressed. Tomatidine has theeffect of increasing and enhancing muscle performance and in maintainingthe weight, especially muscle mass, of an individual.

Staphylococcus aureus is the most pathogenic species of theStaphylococcus genus, responsible for food poisoning, suppurativelocalized infections and physical septicemia (graft, cardiacprostheses). Ogston (1881) coined the genus Staphylococcus to describegrapelike clusters of bacteria (staphylogrape, Gr.) recovered in pusfrom surgical abscesses. The species proves to be an opportunisticpathogen in certain locations or under certain circumstances and isfound in the commensal flora (in 15% to 30% of healthy individuals inthe nasal fossae). S. aureus has pathogenic capacities, in particular aninvasive capacity, a capacity to multiply and to spread in the organism,and also a toxic capacity. S. aureus has a great capacity for developingantibiotic-resistant mutants. In one embodiment, modified Staphylococcusepidermidis is used to produce enhanced amounts of anti-microbialpeptides that inhibit S. aureus biofilm formation, with preferredembodiments employing CRISPR-Cas systems to achieve such modifications.

In various embodiments, due to the inclusion of bacteria-hostileformulations in over-the-counter lotions and related products, the useof conventional lotions is not suggested for employment in conjunctionwith the administration of many embodiments of the present invention.Lotions presently available are believed to be counterproductive to thefostering the beneficial growth of beneficial bacteria on a person'sskin. E.g. salicylic acid is bacteriostatic that limits the growth ofbacteria by interfering with bacterial protein production by downregulating fitness and virulence factor production of bacteria. As it isknown that gram positive and gram negative bacteria prefer slightlybasic conditions pH 7.5 and warm temperatures 37 degrees Celsius (98.6degrees Fahrenheit), the establishment and maintenance of slightlyacidic conditions on one's skin is a preferred objective and is achievedby the fostering of certain bacteria that produce lactic acid on aperson's skin.

All gram negative bacteria are disease producing. As such, one aspect ofthe present invention is directed to reducing the number of gramnegative bacteria on a person's skin by adjusting the overall local pHof the skin tissue region by providing bacterial species that areselected to synergistically grow together and establish a desired pHlevel that discourages the growth of gram negative bacteria on the skin.Caution is called for, however, as the pH should not get too low, asfungi, yeast, and molds prefer acid conditions (pH 5.5-6) at roomtemperature to multiply. In this regard, the pH is preferablymaintained, either by bacterial species producing lactic acid at amountssufficient to achieve such levels, or by other pH adjustment methods, inorder to hinder the growth and progression of pyogenic cocci, sphericalbacteria that cause various suppurative (pus-producing) infections.Included are the Gram-positive cocci Staphylococcus aureus,Streptococcus pyogenes and Streptococcus pneumoniae, and theGram-negative cocci, Neisseria gonorrhoeae and N. meningitidis. In termsof their phylogeny, physiology and genetics, these genera of bacteriaare unrelated to one another. They share a common ecology, however, asparasites of humans. The Gram-positive cocci are the leading pathogensof humans. It is estimated that they produce at least a third of all thebacterial infections of humans, including strep throat, pneumonia,otitis media, meningitis, food poisoning, various skin diseases andsevere types of septic shock. The Gram-negative cocci, notably theneisseriae, cause gonorrhea and meningococcal meningitis. Again, thereduction of virulence factors of such bacteria via CRISPR-Cas or Cpf1systems reduces the incidence of infections caused by such bacteria andleads to methods and systems for establishing and maintaining a healthyskin microbiome, free of disease.

In yet other embodiments, bacteria are modified to express certaincompounds that deter mosquitoes from alighting on an individual's skin.In certain embodiments bacteria are modified to produce amounts of DEET,with such bacteria being contacted to an individual's skin. In stillother embodiments other known insect repellents such as eucalyptol,linalool, and thujone, are expressed by such bacteria to deter insects.In still other embodiments, bacteria are modified to express a proteinmember of the ionotropic receptor family, IR40a, which is a DEETreceptor. In addition, other repellent proteins structurally related toDEET may be employed to repel insects, such as mosquitoes and flies.

One aspect of various embodiments is directed to the expression ofparticular phytochemicals by CRISPR-Cas modified bacteria to amelioratea human disease. Phytochemicals exert their antibacterial activitythrough different mechanisms of action, such as damage to the bacterialmembrane and suppression of virulence factors, including inhibition ofthe activity of enzymes and toxins, and bacterial biofilm formation.These antibacterial effects of phytochemicals may be due to the presenceof one or more of alkaloids, sulfur-containing phytochemicals,terpenoids, and polyphenols and also may involve a synergistic effectwhen used in combination with conventional antibiotics, thus modifyingantibiotic resistance.

Still other aspects of the present invention are directed specificallyto the skin of an individual's scalp, and more particularly with thetreatment of dandruff. Dandruff is an unpleasant scalp disorder commonto human populations. Dandruff is a common scalp disorder that hasoccurred for centuries and has a prevalence of nearly 50% in theworldwide population. The formation of dandruff has been studied fordecades, but no coincident view has been widely accepted. The scalp iscovered with pilosebaceous units and sweat glands. Human sebum is acomplex mixture of triglycerides, squalene, cholesterol esters, waxesters and cholesterols that are secreted from the scalp. The secretionof sebum is controlled by sebaceous gland activity and the sebumsecretion rate increases throughout a person's teenage years, reachesthe highest in the 15- to 35-year-olds and appears to decline in olderadults. Throughout the active period of sebum secretion, the secretionrate is higher in males than in females.

Sebum quantity and water content are negatively correlated with theformation of dandruff. Moreover, a significant relationship existsbetween two reciprocally inhibited bacteria, Propionibacterium andStaphylococcus, on the scalp of individuals. Thus, one aspect of thepresent invention relates to the adjustment of the balance of certainbacteria on an individual's scalp, and specifically, the enhancement ofPropionibacterium and suppression of Staphylococcus, to leads to areduction in dandruff. The host physiological conditions affect themicrobial flora living on the scalp by affecting the scalpmicroenvironment. Sebum is an important food source for the growth ofscalp bacteria and as saturated fatty acids in sebum are consumed,unsaturated fatty acids are left on the skin. Staphylococcus populationson a person's scalp indicate a significant positive correlation withdandruff, while Propionibacterium and Labrys show a significant negativecorrelation with dandruff. Dandruff is therefore associated with thebalance of these two genera. Propionibacterium is affected by variousconditions, including sebum and water content and tends to exist on theside scalp region on the scalps of men. Staphylococcus is present at ahigher ratio on the top region of the scalp and is negatively associatedwith the water content. Propionibacterium can secrete bacteriocins tosuppress the growth of Staphylococcus, whereas Staphylococcus canmediate the fermentation of glycerol and inhibit the overgrowth ofPropionibacterium. Compared with a normal scalp, the dandruff region haddecreased Propionibacterium and increased Staphylococcus. Thus, thebalance between Propionibacterium and Staphylococcus is important to theseverity of dandruff. Scalp sebum acts as a food source forPropionibacterium, and a high water content provides a suitableenvironment for Propionibacterium growth. Adjusting the equilibrium ofthe bacteria, particularly by increasing the Propionibacterium anddecreasing the Staphylococcus on the scalp, lessens the severity ofdandruff. Regulating the physiological conditions is therefore importantto inhibit the development of dandruff.

Other aspects are directed to adjusting the type and amount of bacteriaon a person's body, and in particular in the underarm region, so thatmalodor issues can be addressed. In one embodiment, a deodorantcomposition includes a mixture of bacteria selected to reduce axillaryodor, and specifically involve the promotion of the growth ofStaphylococcus epidermidis bacteria, and the inhibition of the growth ofCorynebacterium striatum bacteria.

Further embodiments of the invention are directed to the employment offungi agents in treating skin conditions. One of the least studiedbiochemical-chemical systems in nature is the relationship existingbetween microorganisms and their plant hosts. Two endophytic fungi,Muscodor albus from Cinnamomum zeylanicum and M. roseus produce amixture of volatile antimicrobials that effectively inhibit and kill awide spectrum of fungi and bacteria. In certain embodiments of thepresent invention, the above fungi and/or the genes providing for theiranti-bacterial characteristics, are employed as part of a skinmicrobiome agent such that particularly undesired bacteria that mightotherwise thrive on an individual's skin, will be killed or reduced inpopulation.

As depicted in FIG. 13 , L. crispatus can be employed in variousbacterial formulations to enhance the health of an individual's skin.Preferably, L. crispatus is included with amounts of other ingredients,including at least one of probiotics, prebiotics, and otherskin-beneficial ingredients. In certain embodiments, at least one, butoften at least two of L. reuteri and/or L. johnsonii and L. crispatusare used in a beneficial bacterial composition for topicaladministration, with the objective being to generate desired amounts ofmetabolites sufficient to reduce inflammation, especially through theproduction of tryptophan metabolites, believed to act as AHR agonists.In one embodiment, live bacterial cells of Lactobacillus crispatus areadministered to the surface of an individual's skin at a dosage of atleast 10⁸ CFU to reduce inflammation through the localized production oftryptophan metabolites. Preferably the skin formulation also includesglycogen and a stimulant for the production of ceramide. The metabolitesproduced by the L. crispatus applied to the kin include indole-3-aceticacid, IAld, Indole-3-Ethanol, Indole-3-pyruvate, and indole-3-aldehyde.Thus, the localized production of tryptophan-derived bacterialmetabolites reduces the amount of inflammation the individual wouldotherwise experience. Preferred topical formulations include acombination of live L. crispatus, prebiotic glycogen, and at least onebarrier-enhancing/moisturizing compound. In certain embodiments L.crispatus is combined with Human milk human milk glycans to facilitatethe generation of beneficial tryptophan metabolites.

In still other embodiments, bacteria are selected for application to anindividual's skin in accordance with an individual's microbiomechronobiology, thereby providing a novel therapeutic approach totreating adverse skin diseases and conditions. Certain aspects of thepresent invention are therefore focused on conditions and/or diseasesinduced by circadian clock disruption that can be mediated by adjustingan individual's microbiome composition and function. Thus, the timing ofapplication of microbiota-based therapeutics, such as pre-, pro-, andpost-biotics, is employed to advance the efficacies of treatments asdescribed herein. An individual's microbial circadian rhythms, due toits diurnal variation, is taken into account when administering the typeof bacterial formulations to a person's skin to arrive at preferredtherapeutic responses. This timing of microbiota-based therapeuticsprovides a unique method for addressing skin conditions and diseases ina fashion previously unappreciated by those of skill in the art, thuspermitting the correction of previously unaddressed dysregulation of anindividual's circadian rhythm associated with aging or chronicillnesses, paying attention to such modifications in terms of thediurnally shifting microbiome. Thus, in one embodiment, a skinformulation containing L. crispatus is applied to an individual's skin(e.g. face tissue) at night so that it remains thereon during thesleeping pattern of the individual. The benefits to the individual'sskin due to such a “night mask” includes the reduction of inflammationthat would otherwise be observed on the skin. For example, as an acnetreatment, application of a L. crispatus formulation as described hereinduring sleep, especially when combined with a glycogen in an amountsufficient to sustain the L. crispatus for at least one hour afterapplication to an individual's skin, results in a noticeable reductionin the inflammation that would otherwise occur due to the acne.

In various other and related embodiments, bacterial formulations asdescribed herein are employed to reduce or prevent the progression of atleast one of scarring (e.g., scar relating to sunburn, bed sore, wound,inflammatory lesion, or burn), crack, fissure, heloma, sebum secretion,skin thickening, wrinkle, sun spot, skin tag, keloid scar, dark patch,stretch mark, spider vein, varicose vein, age spot, cellulitis, or poreappearance in an individual. Still other embodiments focus on the amountand/or a frequency of administration that is sufficient to reduce orprevent the progression of blotchiness or discoloration (e.g., vitiligoor post-inflammatory hyperpigmentation) associated with skin of anindividual. Others relate to the amount and/or a frequency ofadministration sufficient to promote firmness, elasticity, radiance,tone evenness, visual smoothness, hydration, or tactile smoothnessassociated with skin, such as to reduce or prevent the progression of awrinkles, fine lines, or deep furrows. Formulations can be prepared toinclude, in combination, a moisturizer, sunscreen, wrinkle cream,retinoid, alpha-hydroxy acid, antioxidant, tretinoin, glycosaminoglycan(GAG), lactic acid, malic acid, citric acid, tartaric acid,hydroquinone, kojic acid, L-ascorbic acid, licorice extract,N-acetylglucosamine, niacinamide, soy, dermal filler, hyaluronic acid orcalcium hydroxylapatite, botulinum toxin, laser resurfacing procedure,ultrasound therapy, chemical peel, glycolic acid peel, trichloroaceticacid, salicylic acid. Some formulations are administered in conjunctionwith nitrite, nitrate, and/or NO.

Thus, one of skill in the art will appreciate that using the bacterialformulations set forth herein, one can practice a method for reducingthe likelihood of a skin condition that causes skin inflammation, byproviding a bacterial formulation that includes at least one of a livebacteria selected from the group consisting of L. reuteri, L. johnsoniiand L. crispatus, preferably where the bacterial formulation includes atleast one prebiotic having glycogen as a component thereof. The skincondition may be, for example, eczema, atopic dermatitis, acne, allergicinflammation, and skin hypersensitivity. The formulation may also beadministered to an individual's skin as a probiotic bacteriotherapy totreat ultra-violet-induced skin damage.

The bacterial formulation is administered to an individual's skin in anamount sufficient for the bacterial formulation to generate an amount oftryptophan metabolites so as to act as an aryl hydrocarbon receptor(AHR) agonist, thereby reducing inflammation on the individual's skin.The bacterial formulation preferably includes L. crispatus and at leastone of a probiotic, prebiotic, and a skin moisturizer and alsopreferably includes live bacterial cells at a dosage of at least 10⁸CFU. Certain embodiments include in the bacterial formulation astimulant for the production of ceramide. The bacterial formulation isdesigned to generate tryptophan metabolites, such as indole-3-aceticacid, IAld, Indole-3-Ethanol, Indole-3-pyruvate, and indole-3-aldehyde.In certain embodiments, the bacterial formulation is applied to anindividual's skin at night so that it remains thereon during thesleeping pattern of the individual. Moreover, certain embodimentsinclude glycogen in an amount sufficient to sustain the bacteria,preferably L. crispatus, for at least one hour after application to anindividual's skin. In most cases there is a localized production oftryptophan-derived bacterial metabolites by the bacterial formulationthat reduces the amount of inflammation the individual would otherwiseexperience. Formulations may include, e.g. a combination of live L.crispatus, prebiotic glycogen, and at least onebarrier-enhancing/moisturizing compound. Some bacterial formulationsinclude human milk human milk glycans in an amount sufficient for thebacteria to generate tryptophan metabolites. In yet other embodiments,the methods involve the employment of a bacterial formulation that alsoincludes a therapeutically effective amount of a bacterial formulationcomprising Nitrosomonas eutropha, preferably provided in the form of alotion, ointment or gel adapted to be rubbed onto a region of anindividual's skin. One may further administer to the individual's skin aprebiotic that includes a nutrient source for the bacteria in thebacterial formulation that is assimilated by the bacteria. Certainbacterial formulations also include Propionibacterium bacteria, withothers including arabinogalactan, or a glycan unit selected from thegroup consisting of a glucose, a galactose, an arabinose, a mannose, afructose, a xylose, a fucose, and a rhamnose. Still other methodsinvolve administering to the skin an extract derived from a helminthselected from the group consisting of Capillaria hepatica, Dicrocoeliumdendriticum, Ascaris lumbricoides, Enterobius vermicularis, Trichuristrichiura, Ancylostoma duodenale, Necator americanus, Strongyloidesstercoralis, Haemonchus contortus, and Trichinella spiralis. Aparticular method for reducing the likelihood of a skin condition thatcauses skin inflammation in an individual human being is directed to theadministration of a therapeutically effective amount of a bacterialformulation comprising at least one of a live bacteria selected from thegroup consisting of L reuteri, L. johnsonii and L. crispatus, andNitrosomonas eutropha. wherein at least some bacteria in the bacterialformulation have been modified by using a using a clustered regularlyinterspaced short palindromic repeats (CRISPR) CRISPR associated protein(Cas) system or a CRISPR from Prevotella and Francisella 1 (Cpf1) systemto enhance the generation of a tryptophan metabolite.

C. acnes strains are capable of producing several virulence factors thatincrease inflammatory capability. This pathogenic property is believedto be related to infectious mechanisms, such as an ability to formbiofilms and the expression of putative virulence factors capable oftriggering host immune responses or enabling C. acnes to adapt to itsenvironment.

Thus, virulence factors potentially involved in the pathogenicity ofthis bacterium are involved in bacterial attachment to target cells,polysaccharide-based biofilm synthesis, molecular structures mediatinginflammation, and the enzymatic degradation of host tissues. It producesseveral proteins or glycoproteins considered to be active virulencefactors, enabling the bacterium to adapt to the lipophilic environmentof the pilosebaceous unit of the skin, as well as various organs itcolonizes. C. acnes is also involved in the maintenance of healthy skinbut can act as an opportunistic pathogen in various inflammatoryconditions, including acne.

C. acnes is the most abundant bacterium in the skin microbiota, and itsload does not seem to differ between healthy skin and skin affected byacne. One aspect of the present invention is directed to the belief thatinflammatory acne is triggered by an imbalance in the skin microbiotaassociated with the selection of specific types of C. acnes. C. acnesinduces a significant production of reactive oxygen species (ROS) bykeratinocytes, mediated by the cytoplasmic NADPH oxidase. The variousphylotypes of C. acnes have different inflammatory potentials andexpress different putative virulence factors. C. acnes is anopportunistic pathogen of low pathogenicity is mediated by severalmolecular mechanisms, including the production of biofilms and theexpression of virulence factors triggering immune responses in the host.Virulence factor genes have been identified in the C. acnes genome, withsome involved in cell adhesion, whereas others mediate inflammation,tissue invasion/degradation in the host, and the synthesis of capsulepolysaccharides. These putative virulence factor genes encodesialidases, neuraminidases, endoglycoceramidases, adhesins, thermalshock proteins, CAMP factors, lipases/esterases, and lipases. Othergenes of C. acnes encode virulence factors, such as adhesindermatan-sulfate (DsA1 and DsA2), CAMP factors, polyunsaturated fattyacid isomerase, HtaA iron acquisition protein and GehA lipase, and heatshock proteins.

Christie-Atkins-Munch-Petersen (CAMP) factors are toxin proteins thatform pores in host membranes, leading to host tissue damage. C. acneshas at least five CAMP factor genes, CAMP1 to CAMP5. Certain aspects ofthe present invention are directed to the interaction of C. acnes withthe skin microbiota has with respect to its influence on itspathogenicity. In certain embodiments the use of C. acnes strains is asa probiotic option to treat skin disorders.

CAMP1 can trigger an innate immune response when C. acnes comes in closecontact with human (immunocompetent) cells, but such CAMP factors, dueto their pore-forming activity, are believed to be involved in accessingnutrients within the sebaceous follicle microenvironment, withoutcausing excessive damage to intact keratinocytes.

One aspect of certain embodiments of the present invention is directedto enhancing the production by C. acnes to produce short-chain fattyacids as metabolites, including

propionate, acetate, butyrate and valerate. Such SCFA production by C.acnes suppresses the growth of S. aureus and therefore prevents thecolonization of S. aureus on the skin. Such production of SFCAs producedby C. acnes also inhibits biofilm formation by S. epidermidis. Thus,there while there are both health-beneficial as well as -detrimentalroles of C. acnes-produced SFCAs, it is believed that a particularbalance of the amount and the type of SFCAs produced is determinative ofsuch benefits and detriments. In one particular embodiment of thepresent invention, C. acnes is modified via a CRISPR system to producepredominantly butyrate to achieve beneficial skin conditions.

In still other embodiments, CRISPR systems are employed to modify C.acnes to produce increased levels of porphyrins so as to affect othermembers of the skin microbiome.

C. acnes in some embodiments is therefore employed to limit colonizationby more potent pathogens and to positively maintain redox homeostasis onthe skin. Despite its past reputation as being a skin pathogen, C. acnesin certain aspects of the present invention is a beneficial skinbacterium that fulfills important roles for skin homeostasis andprotection.

In certain embodiments, the present invention is directed to the use ofJanthinobacterium, and particularly a modified strain that, via a CRISPRsystem, is modified to reduce or eliminate the presence of at least onevirulence factor of the wild type strains, e.g. vioABCDE violacein, flokformation, and strong biofilm, and type VI secretion system (T6SS). Inone embodiment, a method is provided for treating a skin disorder in asubject in need thereof, that comprises topically administering aformulation comprising an effective amount of probiotic bacteria, ametabolite of probiotic bacteria, and/or a cell lysate of probioticbacteria, wherein the probiotic bacteria are human-derivedJanthinobacterium lividum; and the disorder is associated with thepresence of a topical pathogenic microorganism. Janthinobacterium sp.contain genes as virulence factors.

Certain embodiments of the present invention are directed to a method totopically administer a composition of probiotic strains of bacteria,post-biotic metabolites, or other skin-specific compounds to modulate,restore, and/or support a healthy or normal skin microbiome and skinbarrier microenvironment, defined as populational normal skin barrierfunction, tight junction maintenance, skin hydration defined asfilaggrin production and degradation into sufficient levels of naturalmoisturizing factor (NMF) (defined as >1.50 mmol/g protein), PyrrolidoneCarboxylic Acid (PCA) (defined as >1 mmol/g protein), urocanic acid(UCA) (defined as >0.2 mmol/g protein), and histidine (HIS) (definedas >0.08 mmol/g protein), low or no colonization of pathogenic bacteria,fungi, and other microorganisms (defined as but not limited toMalassezia spp, P. acnes, S. aureus, Kliebsiella pneumoniae,Mycobacterium tuberculosis, Streptococcus pyogenes), colonization ofbeneficial bacteria, and non-visible normal levels of inflammation andimmune activity that cannot be visually identified as inflamed, scaly,boils, or damaged skin. Overall, the described optimal skinmicroenvironment otherwise characterized as a microenvironment that doesnot exhibit clinical representations or symptoms of disruption,including manifestations of atopic dermatitis, psoriasis, ichthyosis,acne, vitiligo, tinea versicolor, and seborrheic dermatitis.

Various embodiments are directed to compositions of at least twoprobiotic strains and/or post-biotics administered in the concentrationof at least 1×108 via AFU counting as either live or heat-killedcombinations representative of genomic diversity found in healthy skinmicrobiomes that are resistant to pathogen colonization, includingbacteria selected from the group consisting of Cutibacterium,Staphylococcus, Corynebacterium, and Micrococcus spp. and otherActinobacteria, Proteobacteria, and Firmicutes. The presence of multiplediverse strains is believed to suppress expression of virulence factorsand modulate metabolism on a population-level and thus, variousembodiments are directed to achieving strain diversity in suchcompositions so as to provide and enable the suppression of S.epidermidis' potential transition to pathogenicity.

Certain acne-associated strains produce higher levels of porphyrinswhich can be regulated by the availability of vitamin B12. Certain S.epidermidis strains produce varying amounts of metabolites of theriboflavin biosynthesis pathway that mediate mucosa-associated invariantT (MAIT) cell activation. Thus, certain aspects of the present inventionare directed to modifying strains of such bacteria, preferably viaCRISPR systems, to affect the regulation of porphyrin production and/orto regulate MAIT cell activation. Still other embodiments are directedselecting or generating strains of C. acnes that produce cutimycin, anantimicrobial that reduces the presence of S. epidermidis, in order toalter the ratio of Cutibacterium:Staphylococcus in order to treat skincancer and conditions affecting the aging of skin.

Certain embodiments are directed to providing compositions of at leasttwo probiotic strains and/or post-biotics administered in theconcentration of at least 1×108 via AFU counting as either live orheat-killed to regulate an individual's skin microbiome ecology byutilizing sebum without producing proinflammatory metabolites orbyproducts. Other embodiments focus on compositions of at least oneprobiotic strain that produces lipases that break down sebum lipids intofatty acids and thus, acidify the surface of the skin. Still otherembodiments are directed to compositions where at least two probioticstrains and/or post biotics are provided that possess broadanti-inflammatory activity, preferably employing strains, preferablymodified via CRISPR systems, to have reduced proinflammatory responseson an individual's skin. Certain compositions of various embodimentsinclude at least one probiotic strain that is protective to tightjunction proteins, thus preserving desired attributes of an individual'sskin barrier. While certain embodiments involve the use of diversestrains, some other embodiments are focused on the employment ofcompositions consisting of single strains that do not upregulate innateimmune response genes, such as CXCL1, CXCL3, CXCL8, CXCL10. In otherembodiments such strains do not upregulate other innate immune responsegenes, including the following: IL10RA, PTGS2, F2RL1, TRIM29, TRAF4,LGALS3, CD55, TRIM8, CASP4, IFNGR1, ADA, NOD1, NOS2, APP, e.g. at1:10,000 dilutions. Still other embodiments are directed to compositionsincluding one or more strains that down-regulate the above referencedpathways.

Alternative embodiments of the present invention are directed to theprovision and use of compositions of single strains or multi strainecologies administered as live or heat-killed post-biotic applicationsin at least AFU defined concentrations of 1×108 cells that do notupregulate cytokine genes including the epithelial alarmin TSLP, andCCL20, VEGFA, IL18, and IL23A, with still other embodiments directed tothe down-regulation of these pathways. Certain compositions include atleast one probiotic strain that regulates filaggrin protein expressionand/or degradation in a manner such that an individual's skin moistureis modified and thus, associated methods permit the regulation of anindividual's skin moisture via the provision of a composition ofselected strains where filaggrin expression is altered as compared towild type strains. Loss of filaggrin results in dry skin and skininflammation with type 3 immune responses. Thus, certain embodiments aredirected to increasing the production of filaggrin to avoid thedevelopment of dry skin. Included within the group of compositions ofthe present invention are those that include at least one probioticstrain in combination with medium chain triglycerides and/or fatty acidsin order to inhibit fungal growth and/or biofilm formation, specificallyof the genus Malassezia.

In certain embodiments, in addition to the provision of beneficialstrains as described herein, compositions and formulations includepostbiotic metabolites, preferably fatty acids produced from sebummetabolism by Cutibacterium. Such compositions are topically applied toskin in order to regulate inflammatory activities of other bacteriafound on an individual's skin, to regulate skin pH to inhibit pathogens,and/or to regulate skin moisture, hydration, and barrier properties.Certain embodiments include not only the strains as described herein,but also formulations that include compounds, lipids, proteins, andnutrients found in cellular debris and that are purified. Preferredembodiments include compounds that are able to cleave sebumtriglycerides so as to free glycerol, such as strains that secrete anextracellular lipase that cleaves sebum triglycerides, thus freeingglycerol. The acidification of an individual's skin employing thestrains and compound formulations as described herein are employed toreduce or prevent the colonization by pathogenic microbes. Otherembodiments are directed to the use of at least one probiotic strain,preferably modified using a CRISPR system, to induce theanti-inflammatory cytokine IL-10 on an individual's skin. As one ofskill in the art will appreciate, various compositions of the presentinvention may contain one of live, heat killed, or post-biotic skinapplications of coagulase-negative Staphylococcus (CoNS) species, e.g.S. epidermidis, S. capitis, S. caprae, S. hominis, S. lugdunensis, andS. haemolyticus, that are able to reduce the abundance of S. aureus onan individual's skin.

Certain embodiments are directed to the modification of C. acnes toincrease the production of propionic acid, which is believed to assistin the maintenance of the acidic pH of healthy skin. Thus, provision ofmodified C. acnes, either via selective propagation or CRISPRtechnology, that has increased production of propionic acid (as comparedto wild type strains) is employed to inhibit the colonization of anindividual's skin by pathogenic microbes.

Certain embodiments of the present invention are directed toadministering a composition topically to an individual's skin thatincludes live or heat-killed coagulase-negative Staphylococcus (CoNS)species at the AFU determined concentration of at least 1×108 cells,which are gram-positive, facultative anaerobes, and that is able toinhibit certain pathogenic bacteria, such as S. aureus strains that arecoagulase positive, or capable of coagulating blood. Preferably the CoNSspecies comprise S. epidermidis, S. capitis, S. caprae, S. hominis, S.lugdunensis, and S. haemolyticus. Preferably, the species of CoNSemployed is able to directly kill S. aureus strains on an individual'sskin by secreting antimicrobial peptides, such as lantibiotics,phenol-soluble modulins (PSM), with even more preferred embodimentsemploying CoNS strains that have been modified via CRISPR systems toincrease its selective killing capabilities and/or in combination withspecific abiotic compounds such as monolaurin or lauric acid to furtherreduce the prevalence of the target organism.

Other embodiments are directed to purposefully interfering withcell-to-cell communications (e.g. quorum sensing) of S. aureus, withpreferred embodiments targeting the accessory gene regulator (agr)system that is believed to be necessary for S. aureus skin infection.Thus, embodiments of the present invention are directed to providingcommensal bacteria in a topical application containing at least two ofthe following actives that are able to kill or inhibit the growth andvirulence of certain pathogenic bacteria; comprised of live bacteria atthe concentration of at least 1×108 cells (AFU), or heat-killed bacteriaat the concentration of at least 1×108 cells (AFU), or post-bioticcompounds, or abiotic antimicrobial compounds such as monolaurin.

While specific embodiments and applications of the present inventionhave been illustrated and described, it is to be understood that theinvention is not limited to the precise configuration and componentsdisclosed herein. Various modifications, changes, and variations whichwill be apparent to those skilled in the art may be made in thearrangement, operation, and details of the methods and systems of thepresent invention disclosed herein without departing from the spirit andscope of the invention. It is important, therefore, that the claims beregarded as including any such equivalent construction insofar as theydo not depart from the spirit and scope of the present invention.

It has been observed by the present inventors that producing Haikuresembles the generation of a patent claim. There is requisitestructure, a need to communicate substance and an ethereal quality ofunderstanding. As one of skill in the art of both biology and haiku willappreciate with respect to skin:

-   -   Within it we are    -   Without it we cannot be    -   Guardian for life.

The foregoing has outlined rather broadly various pertinent andimportant features of various embodiments of the present invention. Suchdescription is, however, not to be considered as limiting the inventionin any way. The invention is capable of other embodiments and of beingpracticed and carried out in various ways which will become obvious tothose skilled in the art who read this specification. Also, it is to beunderstood that the phraseology and terminology employed herein are forthe purpose of description and should not be regarded as limiting of theinvention in any fashion. It is important, therefore, that the claims beregarded as including any such equivalent construction insofar as theydo not depart from the spirit and scope of the present invention.

What is claimed is:
 1. A method of reducing the likelihood of a skincondition comprising acne vulgaris that causes skin inflammation in anindividual human being, said method comprising: administering atherapeutically effective amount of a bacterial formulation comprisingat least one of a live bacteria selected from the group consisting ofLactobacillus reuteri, Lactobacillus johnsonii, Lactobacillus crispatus,Cutibacterium acnes, and Nitrosomonas eutropha, said bacterialformulation comprising a lotion, ointment or gel adapted to be rubbedonto a region of an individual's skin, wherein at least some bacteria inthe bacterial formulation have been modified by using a using aclustered regularly interspaced short palindromic repeats (CRISPR)CRISPR associated protein (Cas) system or a CRISPR from Prevotella andFrancisella 1(Cpf1) system to reduce the production of a virulencefactor by at least one of said live bacteria in said bacterialformulation.
 2. The method as set forth in claim 1, wherein the livebacteria is Cutibacterium acnes.
 3. The method as set forth in claim 2,wherein the Cutibacterium acne has the expression of at least onevirulence factor gene modified using a CRISPR system, said virulencefactors gene selected from the group consisting of virulence factorgenes involved in cell adhesion, mediation of inflammation, tissueinvasion, and the synthesis of capsule polysaccharides.
 4. The method asset forth in claim 2, wherein the Cutibacterium acnes has the expressionof at least one virulence factor gene modified using a CRISPR system,said virulence factors gene selected from the group consisting ofvirulence factor genes that encode sialidases, neuraminidases,endoglycoceramidases, adhesins, thermal shock proteins,Christie-Atkins-Munch-Petersen (CAMP) factors, and lipases.
 5. Themethod as set forth in claim 2, wherein the Cutibacterium acnes has theexpression of at least one virulence factor gene modified using a CRISPRsystem, said virulence factors gene selected from the group consistingof virulence factor genes that encode adhesin dermatan-sulfate (DsA1 andDsA2), CAMP factors, polyunsaturated fatty acid isomerase, HtaA ironacquisition protein and GehA lipase, and heat shock proteins.
 6. Themethod as set forth in claim 2, wherein the Cutibacterium acnes ismodified to enhance the production by C. acnes to produce short-chainfatty acids (SCFA) selected from the group consisting of propionate,acetate, butyrate and valerate.
 7. The method as set forth in claim 6,wherein the SCFA production by C. acnes suppresses the growth ofStaphylococcus aureus in an amount to reduce the colonization of S.aureus on the skin.
 8. The method as set forth in claim 6, wherein theSCFA production inhibits biofilm formation by S. epidermidis.
 9. Themethod as set forth in claim 6, wherein the Cutibacterium acnes ismodified via a CRISPR system to increase production of butyrate toachieve beneficial skin conditions.
 10. The method as set forth in claim2, wherein the Cutibacterium acnes is modified via a CRISPR system toincrease production of butyrate to achieve beneficial skin conditions.11. The method as set forth in claim 2, wherein the Cutibacterium acnesis modified using a CRISPR system to produce increased levels ofporphyrins in an amount sufficient to positively affect other members ofthe skin microbiome.
 12. A method for treating an individual sufferingfrom acne vulgaris, comprising, using a clustered regularly interspacedshort palindromic repeats (CRISPR) CRISPR associated protein (Cas),selectively killing or retarding the growth of a pathogenic bacteriawithin the individual, said pathogenic bacteria selected from the groupconsisting of Staphylococcus aureus; Pseudomonas aeruginosa; Klebsiella;Streptoccocus; Salmonella; Shigella; Mycobacterium tuberculosis;Enterococcus; E coli; Clostridium; Neisseria gonorrhoeae; Acinetobacterbaumannii; and Campylobacter, and enhancing the growth of a beneficialbacteria in the individual selected from the group consisting of C.acnes, Akkermansia, Bacteroides, Bifidobacterium, Fusobacterium,Coprococcus, Lactobacillus, Propionibacterium, Ruminococcus,Veillonella, Prevotella, and Streptococcus bacteria.
 13. The method ofclaim 12, wherein the individual has skin cancer and the method furthercomprises administering to the individual an immune checkpoint inhibitorselected from the group consisting of nivolumab, dostarlimab,pembrolizumab, pidilizumab, AMP-224, AMP-514, STI-A1110, RG-7446,BMS-936559, MEDI-4736, AUR-012 and STI-A1010.
 14. The method of claim12, wherein the individual has skin cancer and the method furthercomprises administering to the individual an immune checkpoint inhibitorthat specifically binds to an immune checkpoint protein selected fromthe group consisting of CTLA4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4,BTLA, KIR, LAG3, TIM-3 and VISTA; and wherein the immune checkpointinhibitor is selected from the group consisting of nivolumab,pembrolizumab, pidilizumab, AMP-224, AMP-514, STI-A1110, TSR-042,RG-7446, BMS-936559, MEDI-4736, AUR-012 and STI-A1010.h.
 15. The methodas set forth in claim 12, wherein said CRISPR Cas system comprises Cas3and is delivered using a bacteriophage.
 16. The method as set forth inclaim 12, wherein using the CRISPR-Cas system, said pathogenic bacteriaare killed while sparing other commensal bacteria.
 17. The method as setforth in claim 12, further comprising administering at least twoprobiotic strains to the individual's skin, said at least two probioticstrains being either live or heat-killed and selected from the groupconsisting of Cutibacterium, Staphylococcus, Corynebacterium,Micrococcus spp., Actinobacteria, Proteobacteria, and Firmicutes. 18.The method as set forth in claim 12, further comprising administering tothe individual's skin at least one probiotic strain that does notupregulate innate immune response genes selected from the groupconsisting of CXCL1, CXCL3, CXCL8, CXCL10.
 19. The method as set forthin claim 12, further comprising administering to the individual's skinat least one probiotic strain that does not upregulate innate immuneresponse genes selected from the group consisting of IL10RA, PTGS2,F2RL1, TRIM29, TRAF4, LGALS3, CD55, TRIMS, CASP4, IFNGR1, ADA, NOD1,NOS2, and APP.
 20. The method as set forth in claim 12, wherein usingCRISPR-Cas systems, said pathogenic bacteria are killed while sparingother commensal bacteria, said commensal bacteria selected from thegroup consisting of C. acnes, Faecalibacterium prausnitzii,Bifidobacterium, Lachnospira, Veillonella, Coprococcus, Akkermansiamuciniphila and Rothia.